A laboratory evaluation of the pathogenicity of Bacillus popilliae var. rhopaea, the agent of milky disease in Rhopaea verreauxi (Coleoptera: Scarabaeidae)

Two methods of infection, i.e., feeding known numbers of spores and rearing larvae in contaminated peat, were used to bioassay the susceptibility of Rhopaea verreauxi to Bacillus popilliae var. rhopaea at 23°C. The susceptibility of the three larval instars was similar as measured by the ID₅₀ and IC₅₀ values. However, within an instar, newly molted larvae were less susceptible than mature larvae when infected by the contaminated peat method. It is suggested that this was due to reduced food intake. The range of ID₅₀ values for all bioassays with R. verreauxi larvae were 1.1 × 10⁷ to 4.0 × 10⁷ spores per larva, and IC₅₀ values were 3.4 × 10⁶ to 5.0 × 10⁷ spores per g of contaminated peat. The slope of the probit line was always low (0.6 to 1.8) except for young first-instar larvae infected by contaminated peat when the slope was 4.0. Disease per se did not affect food intake, though intake was reduced at high doses of contaminated peat. Young larvae often died without developing symptoms but, with increasing age, infected larvae were more likely to develop symptoms. Bioassays with Othnonius batesi and Rhopaea morbillosa indicated a much lower susceptibility per os than for R. verreauxi. It is concluded that the potential for using B. popilliae var. rhopaea to control R. verreauxi is high, but the bacillus is unlikely to be of value in control of O. batesi or R. morbillosa.     

Bioassay of Entomophthora against the spotted alfalfa aphid Therioaphis trifolii f. maculata

A method for the bioassay of Enthomophthora spp. against aphids is described. Twenty-four isolates comprising five species of fungi were screened for activity against Therioaphis trifolii f. maculata. The only isolates with a high level of activity were those of E. sphaerosperma obtained from aphids. The initial bioassays with E. sphaerosperma indicated that aphids, starved for 24 hr during inoculation with E. sphaerosperma primary spores, were less susceptible than those removed from the plants, for just the period of exposure to the primary spore shower. Using the latter procedure, five assays of the most pathogenic isolate gave a mean LC₅₀ of 11.3 primary spores/mm² while bioassays of four other isolates gave LC₅₀ values ranging from 15.7 to 27.3 spores/mm². The potential of E. sphaerosperma as a microbial control agent for T. trifolii f. maculata in Australia is discussed.     

Stored Bacillus popilliae spores and their infectivity against Popillia japonica larvae

Bacillus popilliae spores were stored for about 7 years under three separate conditions: frozen in sterile distilled water, smeared on glass microscope slides, and stored in loam soil at room temperature. In separate experiments, each of the 7-year-old preparations was fed to Popilla japonica larvae at concentrations of 10³, 10⁵, 10⁷, and 10⁹ spores/g of soil. A significant decrease in the percentage of larvae infected occurred in all of the aged spore tests. B. popilliae spores stored in soil, for the extended period, produced 3% larval infection only at the 10⁹ spores concentration; similar results were obtained from frozen spores. When P. japonica larvae were fed spores stored dried on slides, about 20% of the larvae developed milky disease. When aged frozen spores were artificially injected into larvae, 12% became infected at concentrations of 1 × 10⁶ spores/larvae; dried spores at the same concentration infected about 38% of the insect larvae. We conclude from these data that aged B. popilliae spores are significantly less infective against P. japonica larvae than young spores.     

Ecology of Bacillus thuringiensis in storage moths

A disease-free stock of Plodia interpunctella was produced by a continuous rearing technique. In dense populations of this stock, 10⁴ or more spores of H serotype V Bacillus thuringiensis applied at one point on the surface of 200 g of food were required to cause epizootics, compared with 10⁷ or more when spread evenly over the surface. In infected populations, spores contaminated the surfaces of all stages of the insect. In diseased larval cadavers there were 5.6–42.2 × 10⁸ spores/g of dry insect (P. interpunctella, Ephestia cautella, Anagasta kuehniella, Ephestia elutella, and Galleria mellonella). Larvae did not cannibalize live larvae while food was present though they sometimes ate cadavers. This is the most potent means of natural spread of the disease. Occurring mainly in protected situations such as food stores, natural infections are usually light, but occasionally spectacular surface accumulations of dead larvae occur, possibly associated with stress, physiological condition of the larvae, serotype of the bacterium, or behavior pattern such as migration. Natural disease may curb infestations in debris, but it attacks too late to prevent excessive damage to stored food. A prophylactic, even admixture of 2 × 10⁹ spores/200 g of food is required for effective insect control.     

Virulence of entomopathogenic fungi against Culex pipiens: Impact on biomolecules availability and life table parameters

Culex pipiens mosquitoes considered as vectors for many arboviruses such as the West Nile virus and encephalitis virus showing a global impact on human health. The natural management of the aquatic stages of this pest is crucial for maintaining an insecticide-free and sustained environment. The present work focused on studying the biological and biochemical effects of the entomopathogenic fungi: Metarhizium anisopliae, Beauveria bassiana, and Paecilomyces lilicanus, against 3^rd instar larvae of Culex pipiens laboratory colony. The results revealed that M. anisopliae showed maximum larval mortality (88%) with the lowest lethal time (LT₅₀) (22.6 hrs) at 10⁸ spores/ml followed by B. bassiana (73.33%) with LT₅₀ (38.35 hrs), while P. lilicanus showed minimum percent mortality (65%) with highest LT₅₀ (51.5 hrs). The median lethal concentration (LC₅₀) values were found to be 1.027 × 10⁵ spores/ml for M. anisopliae, 1.24 × 10⁶ spores/ml for B. bassiana, while it was 8.453 × 10⁶ spores/ml for P. lilicanus. A reduction in female fecundity, number of hatched eggs, pupation and adult emergence percentage were recorded. The biochemical analysis of the treated larvae revealed different quantitative decrease in total soluble proteins, lipids, and carbohydrate hydrolyzing enzymes compared to control. Histopathological effects of fungal infection upon insect cuticles, muscles, and midgut were investigated. Based on the obtained results, M. anisopliae proved its superior virulent effect as a bio-control agent against Cx. pipiens.     

Solute effects on Lagenidium giganteum: zoospore motility and bioassay reproducibility

The zoospores of Lagenidium giganteum rapidly lose motility when dispersed in deionized water. Several organic solutes were tested for the ability to prolong zoospore activity. Peptone at 0.2 and 0.05 g/liter was more effective than methionine and glucose, individually or in combination. The use of 0.2 g/liter of peptone as a medium for bioassays of L. giganteum against 3-day-old Aedes aegypti reduced the mean LD₅₀ to 12.9 zoospores/ml as compared to 133 with field water and 124 with deionized water. The use of peptone also dramatically improved the reproducibility of the assays and the goodness of fit of the resultant probit regression lines. The mean χ² values were 7.4 for 0.2 g/liter of peptone, 26.8 for field water, and 47.8 for deionized water. It is suggested that the erratic results obtained from use of deionized water are due to variation in the osmotic stress to which the zoospores were exposed, depending on the amount of debris that is introduced into the assays along with the mosquito larvae.     

The susceptibility ofTribolium castaneum [Col.: Tenchrionidae] toFarinocystis tribolii [Protozoa: Schizogregarinida]

The LT₅₀ ofFarinocystis tribolii Weiser to larvae ofTribolium castaneum (Herbst) increased with the age of the insect indicating that older larvae were relatively more tolerant to the infection though there was 100 % mortality ultimately. The adults were less susceptible than larvae and between sexes, females were more susceptible than males. The number of spores produced increased with the stage of the larvae, but there was no variation in the size of spores in the different instars. The LC₅₀ on 20th and 40th day of inoculation were 1.4×10⁷ and 2.1×10⁶ respectively. Mortality-time due toF. tribolii was shorter at 35 °C than at 25 °C. Sporulation occurred earlier at 35 °C than at 25 °C.     

A bioassay of the pathogenicity of Verticillium lecanii conidiospores on the aphid, Macrosiphoniella sanborni

Methods of obtaining uniform batches of conidiospores and stock insects are described together with an assay technique using isolated apterae on leaf discs at virtually 100% relative humidity. Spore suspensions were applied to the apterae in a Buchner funnel and drained off by suction. The weighted mean slope for 11 bioassays was 2.26, and the weighted mean LC₅₀ was 2.33 × 10⁵ spores/ml of suspension with 95% fiducial limits of 1.61 to 3.38 × 10⁵. The within-assay variances were small (mean 0.0081), but in comparison the between-assay variance was large (0.0659) and partly due to a significant lowering of the LC₅₀s with time after the first assay, thought to be caused by an increasing susceptibility of the laboratory stock of aphids. The effects on precision of varying the numbers of replicate assays, doses within an assay, and insects per dosage are illustrated. One four-dose assay will detect differences of about X8 in the LC₅₀s of two batches of spores, duplicating assays X4; and replication 7 times, X2.     

Assessment of oral virulence against Spodoptera litura,acquired by a previously non-pathogenic Metarhizium anisopliae isolate,following integration of a midgut-specific insecticidal toxin

All entomopathogenic fungi infect insects by direct penetration through the cuticle rather than per os through the gut. Genetic transformation can confer fungi with per os virulence. However, unless the recipient isolate is nonpathogenic to the target insect, mortality caused by a transgenic isolate cannot be attributed solely to oral virulence due to the potential for some simultaneous cuticular infection. Here, a Metarhizium anisopliae wild-type isolate (MaWT) nonpathogenic to Spodoptera litura was genetically engineered to provide a transformed isolate (MaVipT31) expressing the insect midgut-specific toxin Vip3Aa1. Toxin expression was confirmed in MaVipT31 hyphae and conidia using Western blotting. Mortality, leaf consumption and body weight of S. litura larvae (instars I–IV) exposed to a range of concentrations of MaWT conidia were not significantly different to controls although the number of conidia ingested by surviving larvae during the bioassay ranged from 2.3 × 10⁵ (instar I) to 8.1 × 10⁶ (instar IV). In contrast, consumption of MaVipT31 conidia caused high mortalities, reduced leaf consumption rates and decreased body weights in all instars evaluated, demonstrating that oral virulence had been acquired by MaVipT31. Larval mortalities were much more dependent on the number of MaVipT31 conidia ingested than the duration of time spent feeding on conidia-treated leaves (r²: 0.83–0.94 for instars I–IV). LC₅₀ and LT₅₀ trends for MaVipT31 estimated by time-concentration-mortality modeling analyses differed greatly amongst the instars. For 50% kill to be achieved, instar I larvae required 3, 4 and 5 days feeding on the leaves bearing 103, 28 and 8 conidia/mm² respectively; instar IV larvae required 6, 7 and 8 days feeding on leaves bearing 1760, 730 and 410 conidia/mm² respectively. Our results provide a deeper insight into the high oral virulence acquired by an engineered isolate and highlight its great potential for biological control.     

The reproduction potentials of four entomopathogenic nematode strains related to cost-effective production for biological control

Bioassays to evaluate the mortality, virulence and reproduction potentials of four indigenous EPN strains, S-PQ16, S-BM12, H-KT3987 and H-CB3452 on insect larvae of mealworm (Tenebrio molitor) and greater wax moth (Galleria mellonella) revealed the highest mortality rates of two insect larvae at the highest inoculation dose of 100 IJs to range from 89 to 100 percent and 94.3–100 percent at 48 h after inoculation, respectively. Virulence was high for all nematode strains, with LC₅₀ values between 29.6 and 47.3 IJs/insect host. The highest IJ yields were different between nematode strains and insect host, from 66.8 × 10³ IJs (S-PQ16) to 118.6 × 10³ IJs (H-KT3987) on T. molitor, and from 54.2 × 10³ IJs (S-BM12) to 163.3 × 10³ IJs (H-KT3987) on G. mellonella. The culturing cost in terms of food expenditure for rearing insect larvae varied between insect larvae and nematode strains, from 6.76 to 26.63 USD per billion IJs for nematode strains cultured on T. molitor larvae and from 3.54 to 7.81 USD per billion IJs for nematode strains cultured on G. mellonella larvae. The full cost for a nematode product of 2.5 × 10⁹ IJs per hectare, produced through in vivo mass culturing, of the most efficient nematode strain, H-KT3987, was 191.3 USD, slightly cheaper than 199.4 USD for the same nematode product produced through in vitro mass culturing.     

Dosage-mortality and time-mortality responses of the armyworm Spodoptera exempta to a nuclear polyhedrosis virus

Third-instar Spodoptera exempta larvae were fed on young maize leaves treated with 20 μl of polyhedral inclusion body (PIB) suspension of concentrations that varied from 1.6 × 10² to 1.6 × 10⁹ PIBs/ml. Daily observations were kept on mortality rates. A probit analysis on the results gave an LD₅₀ value of 48.4 PIBs/larva (lower and upper fiducial limits 39.2 and 59.4 PIBs/larva, respectively), and an LT₅₀ that varied from 146.2 to 221.3 hr, depending on the dosage. LD and LT values obtained show the high pathogenicity of S. exempta nuclear polyhedrosis virus to its host.     

Pathology and Properties of the Tetravirus Helicoverpa armigera Stunt Virus

A quantitative study of the pathogenicity of Helicoverpa armigera stunt virus (HaSV) (Tetraviridae) isolates toward larvae of several heliothine species was conducted along with studies on the stability of the virus to a variety of chemical, enzymic, and temperature treatments. Surface contamination bioassays of several HaSV isolates against H. armigera produced 50% effective concentration (EC₅₀) estimates ranging between 568 and 9244 virus particles (vp)/mm². Against mid 1st instar larvae of H. armigera, H. punctigera, and Heliothis punctifera, EC₅₀ estimates for one isolate were 1288, 16,137, and 2667 vp/mm², respectively. The virulence of HaSV infection varied markedly with the age at which larvae were exposed to the virus. Presentation of the virus to the first three instars of H. armigera was accompanied by cessation of feeding, growth retardation, and eventual lethality, whereas no adverse effects were observed when later instars were exposed to the virus, even at very high concentrations. Active HaSV was recovered from frass of larvae exposed to the virus as 1st instars. Household bleach (1% v/v; 0.04% w/v available chlorine, 0.004% w/v NaOH), formaldehyde (1% w/v), and temperatures ≥65°C completely inactivated HaSV in suspension. Treatments with ether, proteinase K (1 mg/ml), H. armigera gut contents, and temperatures between 22 and 55°C partially inactivated virus activity. No observable inactivation was observed after treatment with chloroform, chymotrypsin (1 mg/ml), trypsin (1 mg/ml), or RNase A (1 mg/ml). The virus was stable between pH 2.8 and pH 10.0 with around 60% loss of activity observed at pH 11.4. The pattern of pathogenic effects seen in several other insect species challenged by high concentrations of HaSV indicated that the host range of the virus is limited to species within the lepidopteran family Noctuidae. The apparently restricted host range of HaSV along with a number of other features indicate that this virus has considerable potential for the development of novel control agents for use against heliothine pests.     

A novel milky disease organism from Australian scarabaeids: Field occurrence,isolation, and infectivity

A novel milky disease organism has been found causing disease in Aphodius tasmaniae and other scarabaeid larvae in the field in Australia. The sporangium is exceptionally long, measuring 10.5 × 1.5 μm, with a small central spore, measuring 1.0 × 0.6 μm. The vegetative cell is about half the size of the sporangium. The disease was easily transmitted by injection of spores into the hemocoel, with typically milky symptoms developing in 2–4 weeks. Spores will form in vivo at temperatures down to 12°C. For A. tasmaniae third-instar larvae, the ID₅₀ by injection was 3 × 10² spores/larva, yet no infection resulted when larvae were reared in peat containing up to 10⁸ spores/g, i.e., the disease was not successfully transmitted per os. All 10 species of scarabaeids tested were susceptible to the disease when spores were injected; however, all attempts to infect larvae per os were unsuccessful. In vitro culture was also unsuccessful.     

Spore Load of Ascosphaera Species on Emerging Adults of the Alfalfa Leafcutting Bee, Megachile rotundata

The spore load of Ascosphaera species spores on larval chalkbrood cadavers and newly emergent adults of the alfalfa leafcutting bee, Megachile rotundata, was determined. The spore content of chalkbrood cadavers ranged from 3 × 10⁶ to 5 × 10⁸. Adults emerging through zero to nine cadavers carried spores on all body parts examined by scanning electron microscopy. Estimates of the total number of spores obtained from a series of adult washes ranged from 9 × 10⁴ to 8 × 10⁷. Some adult males which emerged through no cadavers carried 10⁴ to 10⁵ spores, indicating that nesting materials might also have been contaminated. However, the control of chalkbrood in commercial bee populations may not be accomplished simply by providing clean nesting materials as adults may still emerge through diseased larvae.     

The use of a microinjection procedure for large-scale production of the microsporidian Nosema eurytremae in Pieris brassicae

Nosema eurytremae, a microsporidian parasite of Malaysian trematodes, was injected at the rate of 1 × 10⁴ spores/larva into Pieris brassicae. The larvae, which subsequently pupated, were incubated at 25 to 26°C and on harvesting 19 days later yielded an average of 6 × 10⁸ spores/pupa. This was equivalent to 60,000 times the initial dose. Purity of filtered, washed spore suspensions ranged from 80 to 99% with up to 20% host debris.     

Comparative virulence of Nosema plodiae and Nosema heterosporum in the Indian meal moth, Plodia interpunctella

When larvae of the Indian meal moth, Plodia interpunctella, were fed diets containing spores of Nosema plodiae, the number that survived to the adult stage decreased and the rate of adult emergence was retarded as the concentration of spores was increased; all surviving adults were infected. Also, when larvae were reared on diets containing spores of Nosema heterosporum, the number that survived to the adult stage decreased as the concentration of spores was increased; however, no relationship was apparent between concentration of spores and the rate of adult emergence. The LC₅₀'s of N. plodiae and N. heterosporum were 8.09 × 10⁶ and 4.52 × 10³ spores/g diet, respectively, which confirmed preliminary observations regarding the relative virulence of the two species of Nosema to Indian meal moth larvae.     

The effect of the granulovirus (PapyGV) on larval mortality and feeding behaviour of the Pandemis leafroller,Pandemis pyrusana (Lepidoptera: Tortricidae)

An indigenous betabaculovirus (PapyGV) of the Pandemis leafroller, Pandemis pyrusana (Kearfott), was studied in the laboratory and greenhouse to determine how the virus affected leafroller mortality and foliar damage. Probability of mortality increased with virus concentration as observed after 7 and 10 days of feeding on virus treated diet in neonates and second instar larvae. LC₅₀ estimates for neonates at 7 and 10 days was 2743 and 389 occlusion bodies (OBs)/mm². For second instars, LC₅₀ was 139,487 and 813 OBs/mm² at 7 and 10 days. There was no biologically significant mortality response to increasing virus concentrations by fourth instar larvae; however, when fourth instar larvae were infected with virus on diet and then fed apple leaves, the leaf area consumed declined up to 50% with higher virus concentrations. In a greenhouse study, neonate larvae that fed on seedlings treated with water showed >90% survival and 80% pupation rate of larvae after being transferred to diet. In contrast, larvae that fed on apple seedlings sprayed with 3×10⁶ OBs/ml showed poor survival when transferred to diet after acquiring the virus and failed to reach the pupal stage. This virus shows promise for population regulation and can produce reduction in feeding damage.     

Is It Feasible to Use Optical Brightener Technology with a Baculovirus Bioinsecticide for Resource-Poor Maize Farmers in Mesoamerica?

Stilbene-derived optical brighteners greatly enhance the infectivity of a number of baculoviruses. This technology has been patented for use with insect pathogenic viruses in the United States and Canada. A baculovirus is currently being tested for its potential as a biological insecticide of Spodoptera frugiperda (Lepidoptera: Noctuidae), the principal insect pest of maize in Mesoamerica. A multiply embedded nucleopolyhedrovirus isolate originally from Nicaragua was bioassayed alone and in the presence of the optical brightener Tinopal LPW (1%), using second instar S. frugiperda larvae. The LC₅₀ value of the virus alone was calculated at 82.1 polyhedral inclusion bodies (PIBs)/mm² of diet compared with 0.71 PIBs/mm² in the presence of Tinopal LPW. In contrast to other studies, the mean time to death of larvae exposed to virus and Tinopal LPW was significantly extended compared to larvae inoculated with virus alone. Analysis of the results of eight independent field trials in Mexico and Honduras revealed a significant positive relationship between log virus dose and percentage mortality observed in S. frugiperda larvae. Virus-induced mortality was approximately 50% at the highest application rate tested: 1000 larval equivalents (LE) of virus/ha. When the impact of parasitism was taken into account, larval mortality increased to 45.0–90.7% in plots treated with virus at 250 LE/ha or more. A cost analysis indicated that approximately 60% pest control can be achieved as a conservative estimate with virus application and the action of parasitoids for the price of a chemical insecticide. Formulating the virus with an optical brightener appears to be an attractive option based on laboratory findings but requires field testing. The use of optical brightener technology will probably be feasible for maize growers in Mesoamerica only if it is highly effective at very low concentrations (<0.1%) or the volume of the virus application can be reduced.     

Effects of a novel microsporidium on the black vine weevil, Otiorhynchus sulcatus (F.) (Coleoptera: Curculionidae)

A newly discovered microsporidium infecting the black vine weevil, Otiorhynchus sulcatus (F.) (Coleoptera: Curculionidae), provisionally placed in the genus Canningia, was studied to determine its impact on O. sulcatus. O. sulcatus populations from several locations were sampled and evaluated for microsporidiosis. A very low prevalence of the disease was observed in all locations surveyed (<3.0%). Laboratory studies were conducted by orally exposing both larvae and adults of O. sulcatus to varying concentrations of Canningia sp. spores. Larval bioassays at a variety of dosages (0, 10, etc.) were performed to evaluate pathogen infectivity, larval survival and growth. Adult bioassays (dosages: 0, 10, etc.) were performed to evaluate longevity, fecundity and mechanisms of vertical pathogen transmission. Larvae and adults were infected in all spore treatments. Larval growth was significantly reduced at dosages above 10 spores/larva. Adults infected at all dosages experienced high levels of mortality and fecundity was reduced to zero. Greenhouse trials were performed to determine if larvae feeding in soil acquired infections when spores were topically applied as a drench application (0, 10⁵, 10⁶, 10⁷ spores/pot). Established larvae feeding on plant roots in pots developed infections when exposed to drench treatments of 10⁶ and 10⁷ spores/pot after 14-21 days. Canningia sp. is an acute pathogen of O. sulcatus infective to both larvae and adults. Topically applied spores also infected larvae feeding on roots in soilless potting media, suggesting the possibility of using this pathogen in a microbial control program.     

Foliar application ofNosema pyrausta for suppression of populations of European corn borer

In 1974, an application of the microsporidan,Nosema pyrausta (Paillot, 1927)Kotlan, 1928, with a back-pack type sprayer (22.5×10⁷ spores/plant) to whorl stage maize infested with European corn borers,Ostrinia nubilalis (Hübner) reduced the number of larvae/plant by 48.1% and produced an infection of 15.3×10⁴ spores/mg of larval weight in 62.1% of the collected larvae. In 1975, applications of 24.3×10⁷ spores/plant to similar maize, in 2 separate tests, reduced the number of larvae/plant by 18.8 and 43.8% and caused an infection of 14.3 and 19.1×10⁴ spores/mg of larval weight in 65.9 and 63.3% of the collected larvae. Also, in 1975, applications of 24.3×10⁷ spores/plant to pollen shedding maize in 2 separate tests reduced the number of larvae/plant by 17.2 and 14.1% and caused an infection of 24.3 and 27.2×10⁴ spores/mg of larval weight in 99.2 and 95.2% of the collected larvae.