Inhibition of the accumulation of viral polypeptides in the densonucleosis virus-infected midgut of the silkworm,Bombyx mori,reared at a supraoptimal temperature

Effect of a supraoptimal temperature on the accumulation of viral polypeptides in the midgut was examined by immunoblot analysis in the larvae of the silkworm, Bombyx mori, infected with Bombyx densonucleosis virus type 2. In the larvae reared continuously at 25°C, viral polypeptides were first detected in the midgut at 2 days postinfection (pi) and in the feces at 4 days pi. When the larvae inoculated per os with the virus for 24 hr at 25°C were immediately shifted to 35°C, there were no detectable viral polypeptides in both the midgut and feces throughout the experiment. In the infected larvae shifted from 25° to 35°C at 48 hr pi, viral polypeptides preexisting in the midgut decreased to an undetectable level within 48 hr after the temperature shift, and no viral polypeptides were detected thereafter. Viral polypeptides in the feces of these larvae became detectable at 48 hr (4 days pi) after the temperature shift, as in the larvae at 25°C, and disappeared by 96 hr (6 days pi). These results indicate that a supraoptimal temperature inhibits accumulation of viral polypeptides in the midgut. It is likely that inhibited production of viral polypeptides rather than enhanced discharge of the infected midgut cells is responsible for the inhibited accumulation of viral polypeptides in the midgut at 35°C.     

Seasonal adaptations of populations of the southwestern corn borer, Diatraea grandiosella, from tropical and temperate regions

Seasonal adaptations of populations of the southwestern corn borer, Diatraea grandiosella, obtained from south-central Mexico (19°N latitude) and southeast Missouri (37°N latitude) were compared. Day length and temperature were found to serve as environmental cues to programme the larval diapause of both populations, but different critical values were observed. The critical day length for diapause induction was about 13 hr light/day for Mexican larvae and about 15 hr light/day for Missouri larvae, and was relatively stable at 20 to 30°C. Mexican larvae displayed a less-intense diapause than did Missouri larvae. Some diapausing Mexican larvae maintained at 25 or 30°C pupated in about 15 days, regardless of the day length to which they were exposed. The rate of diapause development of Mexican larvae was high at day lengths between 14 hr and 16 hr, whereas that of Missouri larvae was accelerated at day lengths of 16 hr at 25 and 30°C. Diapause development of Mexican larvae was virtually unaffected by chilling at 10°C, whereas that of Missouri larvae continued at a low rate at 10°C. Selection of Mexican larvae for diapause showed that only four generations were needed to significantly increase the incidence of diapause.     

Effects of temperature and host age upon the encapsulation of Metaphycus stanleyi and Metaphycus helvolus eggs by brown soft scale Coccus hesperidum

Encapsulation of eggs inserted by Metaphycus stanleyi (Hymenoptera: Encyrtidae) into the brown soft scale Coccus hesperidum (Homoptera: Coccidae) became more frequent as the host matured. This occurred with both laboratory reared and field-collected parasites. After parasitism for 24 hr at 27°C, encapsulation frequency did not differ in hosts reared at 20° or at 27°C, but significantly increased in hosts reared at 33°C. When parasitism and rearing were carried out at the same temperature, the percentage of eggs encapsulated increased from 48.7% at 27°C to 94.1% at 33°C. With M. helvolus, the percentage of eggs encapsulated was considerably higher than with M. stanleyi; e.g., 99.3 vs 48.7%, respectively, at 27°C. At 20° and 27°C, some M. helvolus development occurred in the larvae of brown soft scale but none at 33°C; the adult stages of the host encapsulated all the parasite eggs at these temperatures.     

Histopathological effects of Bacillus thuringiensis on the midgut of tobacco hornworm larvae (Manduca sexta): Low doses compared with fasting

The cytology and ultrastructure of the midgut cells of Manduca sexta larvae are described for untreated controls, larvae which fed on a spore preparation of Bacillus thuringiensis, and larvae which were fasted for either 24 or 48 hr. New observations on the ultrastructure of midgut cells in Manduca larvae included the finding of specialized Golgi vesicles in anteriormost columnar cells and of regular arrays of expanded rough endoplasmic reticulum in goblet cells of the posterior midgut region. The present observations reveal that the columnar cells of the midgut responded cytologically in the same way to fasting as they did to exposure to the toxic spores of B. thuringiensis. The goblet cells, however, appeared unaffected by fasting but became swollen in response to feeding of B. thuringiensis spore preparation.     

Comparative laboratory studies on three fungal pathogens of the elm bark beetle Scolytus scolytus: Effect of temperature and humidity on infection by Beauveria bassiana,Metarhizium anisopliae, and Paecilomyces farinosus

Larvae of the elm bark beetle, Scolytus scolytus, were inoculated with conidia of the entomogenous fungi Beauveria bassiana (two strains), Metarhizium anisopliae (two strains), and Paecilomyces farinosus (two strains) and incubated over a range of temperatures (2°, 6°, 10°, 15°, and 20°C). One strain each of B. bassiana and P. farinosus caused infection even at 2°C, whereas the two strains of M. anisopliae caused no infection below 10°C. Infection of adult beetles by B. bassiana (one strain) and M. anisopliae (one strain) was tested at 15°, 20°, and 25°C (B. bassiana) and at 15° and 20°C (M. anisopliae). Fungal infection occurred at all three temperatures, but at 25°C beetles tended to succumb to bacterial infection. The effect of relative humidity on infection of larvae by B. bassiana (one strain), M. anisopliae (one strain), and P. farinosus (one strain) was tested at 51, 74, 86, 90, 95, 97.5, and 100% relative humidity. B. bassiana and M. anisopliae caused some infection at all humidities: with P. farinosus there was no infection at the two lowest humidities. Mortality due to infection by these fungi was most rapid at the highest humidities.     

Effect of temperature on germination,growth, and infectivity of the mosquito pathogen Tolypocladium cylindrosporum (deuteromycotina; hyphomycetes)

The mosquito pathogen Tolypocladium cylindrosporum was examined with regard to its response to temperature. Similar temperature ranges were found for growth, germination, and infectivity of blastospores and conidia. Germination occurred at 8° and 33°C but not at 6° and 35°C. Optimal germination and growth was noted between 24° and 27°C for both spore types. Infectivity of blastospores and conidia at different temperatures was examined by exposing L₂Aedes sierrensis larvae to concentrations of 5 × 10⁵ blastospores/ml or 5 × 10⁶ conidia/ml. Larvae were incubated at 12°, 15°, 25°, and 30°C. Infection occurred at all temperatures tested with LT₅₀ values ranging from 22.7 days (12°C) to 5.6 (25°C) days for conidia and 4.7 days (12°C) to 0.6 day (25°C) for blastospores. These results confirmed earlier findings that blastospores infected and killed host larvae more rapidly than conidia and suggested that this difference is largely due to the more rapid germination rate of blastospores. These experiments demonstrated that T. cylindrosporum can be active against mosquito larvae over a broad range of temperatures encompassing both the cold-water habitat of certain temperate mosquito species as well as the habitat of tropical vector species.     

The effect of temperature on phototaxis and geotaxis by larvae of the crab Rhithropanopeus harrisi (Gould)

Investigations of the effect of sudden temperature change on the phototaxis of Stage I and IV zoeae upon stimulation from horizontal and vertical directions with 500-nm light indicate a temperature-induced geotactic response in larvae of the crab Rhithropanopeus harrisi (Gould). For the horizontal tests both zoea stages were reared at 20 °C. Stage I showed positive phototaxis at temperatures between 15 ° and 35 °C, while Stage IV responded over the range of 10–30 °C. For the vertical tests, larvae, reared at 25 °C, were stimulated with overhead lights. Stage I zoeae ascended at 15 °, 20 ° and 25 °C and descended at 5 °, 10 °, 30 ° and 35 °C. Stage IV zoeae ascended at 20 ° and 25 °C and descended at 5 °, 10 °, 15 °, 30 ° and 35 °C. Although the descent at high temperatures could result from a negative phototaxis, a reversal in phototactic sign at high temperatures was not found in the horizontal experiments and the same vertical movement pattern is observed in total darkness. Upon exposure to high temperatures near the water surface, larvae would descend by means of a positive geotaxis rather than a negative phototaxis. This response involves active swimming by Stage IV larvae and passive sinking by Stage I.     

Cellular immune competence of a Drosophila mutant with neoplastic hematopoietic organs

In the Tum^l mutant of Drosophila melanogaster, the larval hematopoietic organs undergo neoplastic changes and release into circulation large numbers of blood cells. The lamellocytes, and to a lesser extent the plasmatocytes from which they are derived, are the cells that encapsulate various endogenous tissues and form melanotic tumors. The mutation is temperature sensitive, with maximum gene expression manifested at 29°C. The ability of Tum^l larvae to encapsulate eggs of the wasp parasite Leptopilina heterotoma is dependent not only on temperature, with host larvae much more immune reactive at 29°C than at lower temperatures (15° or 21°C), but also on the interval of time following infection when temperature shift experiments are performed. When the shift of parasitized larvae from 21° to 29°C is delayed by 18 hr the hosts are not as immune reactive as those shifted immediately after infection. Since Tum^l larvae are potentially highly immune reactive at the time of infection (with sufficient numbers of lamellocytes in circulation to encapsulate parasites), the low degree of immune competence in hosts shifted to 29°C after 18 hr or maintained at lower temperatures suggests that the increased capacity of blood cells to react against foreign surfaces is dependent on the cells acquiring new or altered recognition and adherence properties at 29°C. The 18-hr delay may provide the parasite with an opportunity to interfere with the acquisition of these specific cellular alterations. Differential hemocyte counts from parasitized larvae show abnormally low lamellocyte counts in susceptible hosts, indicating that successfully developing parasites interfere with the differentiation of hemocytes.     

Development of Bacillus thuringiensis in Galleria mellonella larvae exposed to gamma radiation

A suspension of Bacillus thuringiensis was inoculated at 24 and 72 hr into the oral cavity of Galleria mellonella larvae following exposure to 20, 50, and 70 Kr of gamma radiation, respectively. The cytopathology was conducted after B. thuringiensis had developed for 3, 5, and 7 hr and after radiation damage had developed for 27, 29, 31, 75, 77, and 79 hr in the larvae exposed to 20, 50, and 70 Kr, respectively.B. thuringiensis spores appeared in the midgut lumen from 3 to 7 hr after inoculation of 20 Kr irradiated larvae. At 7 hr after B. thuringiensis infection, and 79 hr after 20 Kr irradiation, the following changes were seen: B. thuringiensis rods appeared adsorbed onto the walls of epithelial cells, a few spores appeared in hemolymph, epithelial cells developed vacuoles, and villi appeared detached from the basement membrane.Within a period ranging from 3 to 5 hr after infection, B. thuringiensis rods attacked vacuolated epithelial cells of most of the 50 and 70 Kr irradiated larvae. At 7 hr after infection and at 31 hr after 70 Kr irradiation, the spores reached the interior of some epithelial cells and were also seen concentrated near the basement membrane.In general, the midgut epithelial cells of the 70 Kr-irradiated groups of larvae appeared highly vacuolated, badly disrupted, and in most cases undistinguishable as a result of attack of B. thuringiensis.In short, B. thuringiensis did not show a characteristic pattern of pathology on 20 and 50 Kr-irradiated midgut cells. The problem of permeability of B. thuringiensis toxin into the irradiated cells needs further investigation.     

Interactions between Bacillus thuringiensis subsp. kurstaki HD-1 and midgut bacteria in larvae of gypsy moth and spruce budworm

We examined interaction between Bacillus thuringiensis subsp. kurstaki HD-1 (Foray 48B) and larval midgut bacteria in two lepidopteran hosts, Lymantria dispar and Choristoneura fumiferana. The pathogen multiplied in either moribund (C. fumiferana) or dead (L. dispar) larvae, regardless of the presence of midgut bacteria. Inoculation of L. dispar resulted in a pronounced proliferation of enteric bacteria, which did not contribute to larval death because B. thuringiensis was able to kill larvae in absence of midgut bacteria. Sterile, aureomycin- or ampicillin-treated larvae were killed in a dose-dependent manner but there was no mortality among larvae treated with the antibiotic cocktail used by [Broderick et al., 2006] and [Broderick et al., 2009]. These results do not support an obligate role of midgut bacteria in insecticidal activity of HD-1. The outcome of experiments on the role of midgut bacteria may be more dependent on which bacterial species are dominant at the time of experimentation than on host species per se. The L. dispar cohorts used in our study had a microflora, that was dominated by Enterococcus and Staphylococcus and lacked Enterobacter. Another factor that can confound experimental results is the disk-feeding method for inoculation, which biases mortality estimates towards the least susceptible portion of the test population.     

Hymenolepis diminuta (Cestoda): Effects of parasite density and temperature on the water balance of Tenebrio molitor and subsequent infectivity of the cysticercoids

The effects of the density of Hymenolepis diminuta and the effects of thermal acclimation on the water balance of Tenebrio molitor were examined. Also, the subsequent infectivity of the cysticercoids for rats were investigated. T. molitor beetles were fed known numbers of H. diminuta eggs and then were kept at 15° or 25°C for 14 days. After 14 days, beetles were desiccated and water loss was determined. Parasite density did not significantly affect transpiratory water loss in T. molitor kept at 15° or 25°C following 24 or 48 hr of desiccation. However, after 72 hr of desiccation, beetles maintained at 15°C evidently could not regulate water efficiently since there was a significant increase in the transpiratory water loss as parasite density increased. Beetles acclimated at 15°C produced fewer cysticercoids than did beetles maintained at 25°C. Also, fewer adult worms were recovered from rats intubated with cysticercoids from heavily infected, 15°C-acclimated beetles. Apparently, heavily infected beetles acclimated to 15°C do not produce viable cysticercoids.     

Selection of indigenous isolates of entomopathogenic soil fungus Metarhizium anisopliae under laboratory conditions

Eight native isolates of the entomopathogenic fungus Metarhizium anisopliae (Metschnikoff) Sorokin were obtained by monitoring soils cultivated in a conventional manner. These isolates were compared in three areas: (a) conidial germination, (b) radial growth and sporulation and (c) ability of conidia to infect Tenebrio molitor larvae. All bioassays were carried out at constant temperatures of 10, 15, and 20 °C. Conidia of individual isolates demonstrated differences in germination after a 24-h long incubation at all evaluated temperatures. At 20 °C, the germination ranged from 67 to 100 % and at 15 °C from 5.33 to 46.67 %. At 10 °C, no germination was observed after 24 h; nevertheless, it was 8.67–44.67 % after 48 h. In terms of radial growth, the culture diameters and the associated production of spores of all isolates increased with increasing temperature. At 10 °C, sporulation was observed in three isolates while all remaining cultures appeared sterile. Three weeks post-inoculation, conidia of all assessed isolates caused 100 % cumulative mortality of treated larvae of T. molitor at 15 and 20 °C with the exception of isolate 110108 that induced 81.33 % mortality at 15 °C. At 10 °C, larval cumulative mortality ranged from 6.67 to 85.33 % depending on the isolate. Isolates 110108 and 110111 showed significantly slower outset and a much lower rate of infection at all temperatures compared to other tested isolates of M. anisopliae. The bioassays were carried out with the purpose to sort and select indigenous isolates of M. anisopliae useful as biocontrol agents in their original habitat.     

Diapause in the mite Metaseiulus occidentalis: Stages sensitive to photoperiodic induction

Stages of Metaseiulus occidentalis sensitive to photoperiod induction of diapause were determined by transferring various stadia into diapause-inducing conditions, and rearing them until adult females could be scored for reproductive condition. When eggs were transferred to 10 hr light at 19°C from 24 hr light at 25°C and the mites reared to adults, 92 per cent entered diapause. When larvae and all subsequent stages were kept under the inductive conditions, 62 per cent of adult females diapaused. Mites transferred as protonymphs into inductive conditions yielded only 10 per cent in diapause, and mites transferred as deutonymphs or newly emerged females did not enter diapause.However, adult females reared from eggs at 19°C under 12 hr light (which is near the critical photophase of 11·2 hr at 19°C) showed an unexpected sensitivity to photoperiod. Some newly emerged females oviposited upon transfer to an 8 hr photophase at 19°C. Some then stopped ovipositing and apparently entered diapause; these females resumed ovipositing after intervals ranging from 34 to 100 days. This was termed ‘switching’ into diapause. Some females reared under a 16 hr photophase at 19°C ‘switched’ also upon transfer as adults to shorter photophases—either 8 or 12 hr at 19°C. Thus, ‘switching’ may be due to transfer to shorter photophases. Promptness of mating vs delayed mating allowed ‘switching’ to be more easily detected.     

Pathogenicity of entomopathogenic hyphomycetes, Paecilomyces fumoso-roseus and Nomuraea rileyi, to eggs of noctuids, Mamestra brassicae and Spodoptera littoralis

Bioassays were conducted to determine the susceptibility of egg masses of Mamestra brassicae and Spodoptera littoralis to different spore doses of Paecilomyces fumoso-roseus and Nomuraea rileyi at 20° and 25°C. P. fumoso-roseus was highly virulent against eggs, whereas N. rileyi provoked only a deferred mortality of larvae hatched from treated eggs. Nevertheless, larval mortality of S. littoralis caused by N. rileyi at 25°C was more effective after first-instar larval contamination than after egg mass treatment. The duration of the egg stage could explain differences of susceptibility between the two noctuids at 25°C. Scanning electron microscopical observations suggested two ways of contamination of newly hatched larvae. First, fungal germinations on the chorion surface suggested that newly hatched larvae might be infected by penetration of the egg integument before hatching. Second, conidia on the egg cuticle could be an entomopathogenic inoculum for newly emerging larvae which fed upon chorions. Results showed that pathogenicity of Hyphomycetes to noctuid eggs might be a promising area of investigation for biological control.     

Diapause induction in the oblique-banded leafroller Choristoneura rosaceana (Lepidoptera: Tortricidae): Role of photoperiod and temperature

Induction of diapause in the larval stage of the oblique-banded leafroller, Choristoneura rosaceana (Harris), was found to be dependent on both photoperiod and temperature. At constant temperatures of 24, 20 and 16°C, short photoperiods induced diapause. The critical photoperiod was between 14–15 h of light per day at 20 and 16°C. At 14 h light: 10 h dark, all larvae expressed diapause. Temperature had a modifying effect, and slightly shifted the larval response to diapause-inducing photoperiods. High constant temperatures of 28°C and above induced diapause in some individuals (< 20%), while fluctuating temperatures of 32 and 16°C in a 12-h cycle resulted in 67% diapause induction, suggesting that diapause could also be induced by fluctuating temperatures, particularly if the higher temperature exceeds 25°C.The first- and the second-instar larvae were the only two stages sensitive to diapause induction. Exposure of adult, egg and third, fourth, and fifth-larval instars to diapause-inducing conditions did not produce diapause. Although diapause was induced in the first or the second instars, it was always expressed in the third or fourth instar.     

Susceptibility of the spruce budworm, Choristoneura fumiferana, and the white-marked tussock moth, Orgyia leucostigmata, to Bacillus thuringiensis: Chemical insecticide combinations

Bacillus thuringiensis mixed with the organophosphate insecticides, fenitrothion (Sumithion), Gardona^®, and Orthene^®, or the synthetic pyrethroid, SBP 1382, was incorporated into synthetic diet and fed larvae of the spruce budworm, Choristoneura fumiferana, and the white-marked tussock moth, Orgyia leucostigma. Mortality was highest when larvae were fed combinations of low concentrations of the insecticides and low to moderate concentrations of the pathogen. The data indicated that applications of a B. thuringiensis dosage expected to produce about 45% mortality of third and fourth instar larvae of the spruce budworm combined with a dosage of fenitrothion causing about 40% mortality or a dosage of Orthene causing from 5 to 25% mortality should result in low budworm survival. With a B. thuringiensis dosage causing 20–60% mortality combined with a fenitrothion dosage causing 15–50% mortality or a sublethal dosage of Gardona, a low survival rate of young white-marked tussock moth larvae may be expected.     

Susceptibility of Heliothis zea larvae to Nomuraea rileyi at various temperatures

Laboratory studies were conducted to determine the susceptibility of various larval instars of Heliothis zea to different spore doses of Nomuraea rileyi at constant and variable temperatures. The fungus was most effective at 20° and 25°C, with a mortality of 80% and 71%, respectively. At 15°C the disease progressed very slowly with larval mortality occurring in 12–28 days post-treatment. Conversely, at temperature ranges above 15°C, the mortality of the larvae occurred in 6–12 days. Three different combinations of variable temperatures included 20–30°, 25–30°, and 20–35°C, but mortality did not exceed 46%. Larvae in the third to fifth instars were more susceptible to infection than were those in the first and second instars.     

Virulence of entomopathogenic Fungi (Fungi imperfecti) for the adults of Acanthoscelides obtectus (Coleoptera: Bruchidae)

Tests with the bean weevil, Acanthoscelides obtectus, in which the hosts were exposed indirectly to various dilutions of conidia of four entomopathogenic fungi showed that mortality was a function of the concentration of the inoculum. In these tests a given spore suspension was sprayed on the internal surfaces of a Petri dish. Adult weevils of a known age were placed in the dish, held there for 24 hr, then removed and kept at 20°C. After 20 days, the host mortality was determined. From the data obtained, it was possible to trace a probit regression line of the mortality in relation to the increasing spore concentration. Infection was observed in hosts exposed to a concentration of approximately 5 × 10⁶ spores/ml up to a maximum of about 1 × 10⁹ spores/ml. The A. obtectus was susceptible to infection by spores of Beauveria bassiana, B. tenella, Metarrhizium anisopliae, and Paecilomyces fumoso-roseus.     

Haemolymphal activation of protyrosinase and the site of synthesis of haemolymph protyrosinase in larvae of the fleshfly, Sarcophaga barbata

When whole blood from 5 day third instar larvae of the fleshfly, Sarcophaga barbata was incubated under nitrogen at 25°C for 16 hr in the presence of salivary glands there was an increase in its protyrosinase content, which amounted to 53% of that which occurs in vivo over the same period. The protyrosinase in ammonium sulphate fractions of haemolymph that were allowed to stand at 4°C for 24 hr following the incubation at 25°C was found to have autoactivated. Analysis of all these fractions revealed the presence of a protyrosinase activator in the 30% saturated ammonium sulphate fraction. When proenzyme and haemolymph activator were mixed there followed a lag period before the rapid phase of activation, the duration of the lag being dependent upon the concentration of both proenzyme and activator. The final activity attained was dependent upon the concentration of proenzyme, but was independent of the activator concentration and was comparable to that obtained using the cuticle activator. The level of activator in the haemolymph increased as larvae aged from 4 to 7 days.The effect of several compounds on the catecholase activity of the activated haemolymph protyrosinase and on the cuticle enzyme is reported and the significance of haemolymphal activation of protyrosinase is discussed.     

Diapause termination in two species of damselflies

Larvae of two species of damselflies, Enallagma hageni and E. aspersum, were collected in southwestern North Carolina and subjected to different combinations of daylength (SD, 11 hr day; LD, 14 hr day) and temperature to determine the factor critical in the termination of diapause. Diapausing larvae were collected in September, and 15 comparable groups of each species were given pretreatments (SD, 10°C; LD, 10°C; or SD, 21°C) for 2, 4, or 8 weeks and then transferred to SD, 21°C or LD, 21°C until emergence. Control groups were maintained under both photoperiods at 21°C.Response times (days from collection to emergence) for both species showed that rapid development required transfer to LD, 21°C regardless of the type of pretreatment or length of exposure. Larvae transferred to LD, 21°C after exposure to any of the three types of pretreatment for equal lengths of time developed at similar rates. Pretreatments at 10°C, which were equally effective with SD or LD, stimulated subsequent rapid development at LD, 21°C to a greater extent than continuous exposure to LD, 21°C conditions, and the longer the exposure to 10°C, the faster the subsequent response. Pretreatments at SD, 21°C also resulted in rapid development, similar to that of larvae exposed to 10°C, upon transfer to LD, 21°C conditions. Long daylengths administered only during pretreatments did not effect rapid development, since all larvae responded slowly when transferred to SD, 21°C. Diapause termination, therefore, was effected by LD, 21°C conditions preceded by exposure to either low temperatures, during which the photoperiod was not important, or short daylengths at 21°C.