A microsporidian pathogen of the poroporo stem borer,Sceliodes cordalis (Dbld) (Lepidoptera: Pyralidae): Effects on adult reproductive success

Larvae of Sceliodes cordalis were infected over a range of ages and doses with Nosema sp. (NSC). The resulting spore loads in adults ranged from 9.3 × 10⁵ to 8.9 × 10⁷ spores and varied with the age of the larva at infection and with the dose ingested. Infection caused small reductions in female weight, mating success, potential fecundity, longevity, and egg viability. The magnitude of spore loads in female moths was not found to influence any of these factors apart from potential fecundity in very low weight moths. The cumulative effects of infection reduced natality in the laboratory colony from 11,400 to 6000 hatching eggs per 100 females. Laboratory reared, infected females were smaller, had a lower longevity, a reduced spore load, and were more fecund (eggs per milligram adult weight) than infected females from a field population. Manipulation of NSC in a biological control program is not considered to be of any value.     

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.     

Mass production and storage of Vairimorpha necatrix (protozoa: Microsporida)

Mass production and storage methods were evaluated for maximization of spores of Vairimorpha necatrix, a promising protozoan for microbial control due to its virulence and prolificity in lepidopterous pests. In vivo spore production was at a maximum when 3rd instar Heliothis zea were exposed to 6.6 spores/mm² of artificial diet surface and reared for 15 days. Approximately 1.67 × 10¹⁰ spores/larva were produced, or ca. 1 × 10¹⁰ spores/larva after partial purification of the spores by homogenization of the larvae in water, filtration, and centrifugation. The spores were inactivated by relatively short exposures to several chemicals which were tested to counteract contamination of the diet surface by fungi in the spore inoculum. Spores of V. necatrix were stored at refrigerated and freezing temperatures for up to 2 years and bioassayed periodically with 2nd instar H. zea. Spores lost little infectivity after 23 months at 6°C if they were stored in a purified water suspension plus antibiotic, but they were noninfective after 18 months at 6°C if stored in host tissue. Storage at ?15°C caused little loss of infectivity whether the spores were stored in water and glycerine, in host tissue, or after lyophilization. The spores withstood lyophilization in host cadavers better than in purified water suspension. Samples of a dry V. necatrix-corn meal formulation, which was prepared for field efficacy tests and stored at ?15° and 6°C, were highly infective after 9 months. Large numbers of V. necatrix spores can thus be produced and later made available for microbial control field trials with little loss of infectivity.     

The differential mortality at various life stages of the greenhouse whitefly,Trialeurodes vaporariorum (Homoptera: Aleyrodidae), by infection with the fungus Aschersonia aleyrodis (Deuteromycotina: Coelomycetes)

The parasitoid Encarsia formosa is commonly applied to control the greenhouse whitefly Trialeurodes vaporariorum in glasshouse tomatoes and cucumbers. Nevertheless, in some cases the control capacity of this natural enemy is insufficient and an additional selective pest-suppressing agent is desirable. The entomopathogenic fungus Aschersonia aleyrodis was applied to cucumber plants carrying whiteflies in different developmental stages. After spraying each leaf with 2 ml of spore suspension (4 × 10⁶ spores/ml) the plants were kept at 100% RH for 24 hr; thereafter the humidity was lowered to 70% RH at 20°C and the photoperiod was 16 hr. Treated eggs did not become infected, but larvae that hatched from these eggs and settled on the treated abaxial leaf surface were infected at the same rate and to the same degree as treated first instar larvae. This suggests that the spores persist for at least 7 days. The final percentages of infection over all instars when treated as young eggs, old eggs, and first larval instars were 94, 93, and 90%, respectively. The final percentages of infection when treated as third and fourth larval instars and prepupae were 76, 28, and 12%, respectively. The older instars were less susceptible and adults were seldom infected by the fungus. Several applications of A. aleyrodis as a microbial insecticide are needed to achieve sufficient control of whitefly populations in glasshouses.     

Transmission and infectivity of spores of Burenella dimorpha (Microsporida: Burenellidae)

Burenella dimorpha infects the tropical fire ant, Solenopsis geminata, producing two morphologically distinct types of spores. A binucleate, nonpansporoblast membrane-bounded (NPMB) spore develops in and destroys the hypodermis, rupturing the cuticle in the pupal stage. A uninucleate, pansporoblast membrane-bounded (PMB) spore develops in the fat body. Adult ants cannibalize ruptured pupae but do not ingest spores. Instead, the spores and particulate foods are diverted to the infrabuccal cavity, formed into an infrabuccal pellet, and fed to fourth-instar larvae only. This larval instar is the only stage in the life cycle of S. geminata that is vulnerable to infection. NPMB spores are infective, but PMB spores do not extrude their polar filaments in the larval gut and are expelled in the meconium upon pupation.     

Effect of Host Plant on the Infectivity of Sl MNPV to Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) Larvae

The susceptibility of Spodoptera litura to SlMNPV infection was markedly affected by phyto-chemicals ingested during the acquisition of viral inoculum on foliage of tomato and cauliflower. The LD₅₀ values computed for second, third and fourth instar larvae assayed on tomato leaves were 254, 819 and 23395 PIBs/larva, respectively whereas, it was 326, 1719 and 43843 PIBs/larva for respective instars when assayed on cauliflower leaves. Thus LD₅₀ values for second, third and fourth instar larvae were 1.28-, 2.09- and 1.87- fold lower, respectively in tomato leaves. Similarly, LT₅₀ values for second, third and fourth instar larvae assayed on tomato leaves were 7.1 and 7.5 days, respectively at inoculum dose of 2.7×10⁴ PIBs/larva whereas, it was 7.7 and 8.0 days for respective instars when assayed on cauliflower leaves at same inoculum. This result also showed that the S. litura were more susceptible on tomato leaves in comparison to cauliflower leaves as the time required for mortality was lower in tomato leaves. The possible biochemical bases for differential level of mortality of S. litura larvae on tomato and cauliflower crops needs to be investigated.     

Selection of Antonospora locustae (Protozoa: Microsporidae) with higher virulence against Locusta migratoria manilensis (Orthoptera:Acrididae)

Antonospora locustae is a microsporidian parasite of grasshopper insects that is used as a biological control agent. We report on laboratory selection of isolates from different regions with increased virulence. Bioassays were conducted against third instar nymphs of Locusta migratoria manilensis. AL2008L01 was originally imported from the USA in 1986, AL2008M01 was isolated from Melanoplus differentialis in USA and AL2008F01 was isolated from infected Fruhstorferiola tonkinensi collected in Guangdong, China. The results showed that all three isolates can infect the locust and that pathogenicity increased gradually with increased dose. The LD₅₀ values of the original isolates at the highest dose (5×10⁶ spores/nymph) were 19, 23 and 22 days and LD₅₀ values were 3.2×10⁵, 3.4×10⁶ and 0.7×10⁶ spores/g, respectively. After selecting for three generations, the virulence of all isolates increased significantly. LT_50s were reduced to 17, 20 and 21 days at the highest dose (5×10⁶ spores/nymph) and LD_50s were reduced to 1.4×10⁵, 2.5×10⁵ and 1.7×10⁵ spores/g.     

Response of Lymantria dispar L. (Lepidoptera: Lymantriidae) to Bacillus thuringiensis subsp. kurstaki at different ingested doses and temperatures

We examined mortality and feeding inhibition response of Lymantria dispar L. (Lepidoptera: Lymantriidae) larvae to ingested doses of Bacillus thuringiensis subsp. kurstaki as a function of dose, instar and temperature. We developed generalized (logistic) linear mixed models and a mixture survival model, commonly used in medical statistics, to analyze the complex data set. We conducted bioassays of Foray 48B with larvae from the NJSS laboratory stock, using droplet imbibing or force-feeding to ensure dose ingestion. The dose causing mortality in 50% of the test population (LD₅₀) under standard test conditions (22 °C) ranged from 0.019 International Units (IU)/larva for first instar larvae (L₁) to 1.6 IU/larva for L₄. Temperature affected larval mortality in two ways. Mortality occurred sooner and progressed more rapidly with increasing temperature (13-25 °C) at each dose level and instar, while the maximum level of mortality attained by each instar decreased with increasing rearing temperature. The mechanisms underlying this effect are being investigated. Larvae that survived exposure to B. thuringiensis resumed feeding after a period that was dependent on instar, dose, and temperature. The equations describing observed mortality and feeding recovery responses were used to construct a simulation model, which was able to predict both processes, and which forms the basis for a process-oriented model that can be used as a decision support tool in aerial sprays.     

Laboratory and field evaluations of the susceptibility of immature Choristoneura rosaceana (Lepidoptera: Tortricidae) to Beauveria bassiana (Hypocreales: Cordycipitaceae)

Abstract

The susceptibility of immature Choristoneura rosaceana (Harris) to Beauveria bassiana-GHA (BotaniGard® 22WP) was evaluated under laboratory and field conditions. Although egg masses ≤24 h old were susceptible to infection by topically sprayed B. bassiana spores in the laboratory and resulting mycosis significantly reduced the percent of neonates able to emerge, no significant egg mass infections resulted from orchard treatment of ≤24–48 h eggs. Exposure to high levels of the B. bassiana product on apple leaf surfaces in the laboratory caused significant dose-related mortality in first to fourth instar C. rosaceana. First instar C. rosaceana were the most susceptible of the larval stages assayed in the laboratory although only 36% of neonates introduced into the orchard prior to Beauveria treatment and 27% of the neonates emerging from orchard treated egg masses became infected. Fourth instar mortalities were similar but moderate when treated with 1×10⁸ spores mL^?1 in the orchard and on leaf surfaces in the laboratory. Beauveria bassiana-induced larval mortalities were significantly higher when the spores were applied directly to the larval integument as opposed to the leaf surface where the insect would encounter the pathogen in a treated orchard. Feeding of C. rosaceana larvae on antibiotic containing meridic diet prior to their use in trials did not impact the susceptibility of the larvae to B. bassiana. Antibiotic containing meridic diet significantly reduced larval C. rosaceana mortality when treated larvae or the spores were placed directly on the diet as opposed to leaf tissue.     

Soybean leaf consumption by Nomuraea rileyi (Fungi: Deuteromycotina)-infected Plathypena scabra (Lepidoptera: Noctuidae) larvae

To determine the impact of Nomuraea rileyi on consumption by the green cloverworm, Plathypena scabra, larvae were reared from eggs obtained from field-collected moths, inoculated with conidia, and placed individually in separate plastic vials with a piece of surface-sterilized soybean leaflet. No significant differences in consumption rates were found between N. rileyi-inoculated and control larvae until after 144 hr post-treatment. After this period, consumption by larvae inoculated as first, second, third, or fourth instars was significantly less than that of control larvae because of mortality. First or second instars inoculated with N. rileyi conidia had significantly longer subsequent stadia than did control larvae. The average LT₅₀ of all inoculated instar groups was 6.5 days, and there was little evidence of significantly different N. rileyi susceptibility among instar groups. In general during the N. rileyi infection period leading to their deaths, inoculated P. scabra larvae had consumption patterns that were very similar to those of healthy larvae.     

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.     

Determination of coleopteran insects associated with spore dispersal of Cryptoporus volvatus (Polyporaceae: Basidiomycota) in Korea

The veiled polypore, Cryptoporus volvatus, is distributed widely in North America and East Asia and is believed to have a mutualistic relationship with coleopteran species—the fungus providing food and shelter in basidiocarps and beetle helping disperse spores.Seventy fresh basidiocarps of C. volvatus were collected from the Japanese red pine (Pinus densiflora) in the spring season of 2013 from two sites in Korea. A total of 251 insects (101 adult and 150 larvae) were collected from the inside of basidiocarps and identified using morphology and mitochondrial cytochrome c oxidase subunit I (COI) sequences. Six species belonging to five coleopteran families were identified. The number of spores attached to the bodies of adult insects was counted and average spore counts for each of the six species ranged between 1.0 × 10⁴ and 5.2 × 10⁵ spores/individual. Across localities, three species were shared (Aethina suturalis, Trogossita japonica and Parabolitophagus felix) and carried spores at high densities on their bodies, making them more likely to aid in spore dispersal.     

Some factors affecting spore replication of Nosema algerae (Microspora,Nosematidae) in Pieris brassicae (Lepidoptera)

The production of Nosema algerae spores was examined in Pieris brassicae. Spore replication in the insect host followed a logistic pattern of development. The factors studied which affected spore production and replication were dose level (5 × 10², 5 × 10³, and 5 × 10⁴ spores per insect), larval instar (fourth and fifth), and cool pretreatment of the insects at 20°C prior to inoculation compared with a constant temperature of 26°C. A three-way analysis showed the interactions between these factors. The logistic pattern of spore replication was used to explain the results.     

Functional Response of Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae) to Sugarcane Whitefly Aleurolobus barodensis (Maskell) in Laboratory Conditions

A functional response study of Chrysoperla carnea (Stephens) larvae to different densities of sugar cane whitefly Aleurolobus barodensis (Maskell) was conducted in test tubes at 26?±?2 °C, 65?±?5 % RH. Chrysoperla carnea showed two different types of functional response in larval instars. First instar exhibits type II. However, second and third larval instars revealed type III functional response. Based on modified Holling’s disk equation, the highest searching rates (a) of 0.82?±?0.0247 h^?1 was found for first instar larva. For second and third larval instars, the attack coefficient (b) were 0.002?±?0.030 and 0.0025?±?0.0424 respectively. The shortest handling time (T_h) per prey was observed at third instar stage (1.574?±?0.0568 h) followed by second and first instar with 1.72?±?0.0411 h and 1.919?±?0.0568 h respectively.     

Isolation and pathogenicity of some South African entomopathogenic fungi (Ascomycota) against eggs and larvae of Cydia pomonella (Lepidoptera: Tortricidae)

Virulent entomopathogenic fungal strains against Cydia pomonella (Lepidoptera: Tortricidae) were isolated and identified in the Western Cape Province of South Africa. Thirty-nine isolates belonging to six species were obtained using the insect bait method. Generally, Metarhizium robertsii (Ascomycota: Hypocreales) was the most frequently encountered species representing 51% of the total number of isolates collected from the soil samples. This is the first report of M. robertsii from southern Africa. Mortality data from an immersion bioassay indicated that the 39 fungal isolates were pathogenic against fifth instar larvae of C. pomonella inducing 47–85% insect mortalities. Two M. robertsii strains, MTL151 and GW461, induced 85% larval mortality and were selected for further evaluations. The exposure of freshly laid eggs to wax papers that were pre-treated with fungal spores ranging from 10³ to 10⁸ spores ml^?1 of MTL151 and GW461 resulted in a significant reduction of egg hatchabilities, from 95 to 66% and 93 to 71%, respectively as spore concentration increased. First instar larval neonates were exposed to apples that were pre-sprayed topically with varied conidia suspensions (10³?10⁸ spores ml^?1). The mean percentage of participating apples with larvae in their cores/flesh significantly reduced from 53 to 10% (MTL151) and 76 to 10% (GW461) of 10 apples, and a concurrent decrease in the incidence of apple fruit rot was observed as conidia concentration increased. Up to 90% of apples treated with 1 × 10⁸ spores ml^?1 had no larvae present in their cores and this result compared favourably with the commercial pesticide Fruitfly®, containing the active ingredient cypermethrin (20 g/l) used at a recommended dose of 0.25 ml/250 ml of water.     

Effect of prolonged storage of spores on field applications of Nosema locustae (Microsporida: Nosematidae) against grasshoppers

Spores of Nosema locustae that were freshly prepared, stored in water at ?10°C for 8 months to 3 years, or stored in cadavers for 1 year at ?10°C were applied on bran at the rate of 10⁹ spores/1.5 lb bran per acre. Applications of fresh spores resulted in higher density reductions and higher incidence of infection among survivors than applications of spores stored in water or cadavers in both a complex of grasshoppers predominated by a single species and in a mixed species complex. Density reductions due to treatment with fresh spores were similar in the two populations, but the mixed species complex had a lower incidence of infection than the complex in which one species predominated. Applications of fresh spores reduced grasshopper densities in both complexes to levels below the economic thresholds.     

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.     

Pathogenesis of Bacillus sphaericus strain SSII-1 infections in Culex pipiens quinquefasciatus ( = C. pipiens fatigans) larvae

Numbers of viable bacteria in second instar Culex pipiens quinquefasciatus larvae were determined following ingestion of pathogenic strain SSII-1 and nonpathogenic Bacillus sphaericus. Numbers of nonpathogenic B. sphaericus recovered from larvae declined rapidly after cessation of feeding, as did numbers of pathogenic SSII-1 cells fed at LD₂₀ dosage. When pathogenic cells were fed at LD₇₀ dosage, the number of B. sphaericus in larvae increased following initial decline. When chloroformtreated SSII-1 cultures, in which all bacteria except spores were dead, were fed at LD₁₀ and LD₉₈ dosages, no viable B. sphaericus were recovered from larvae. In all SSII-1 treatments, other bacterial flora multiplied rapidly in larvae following onset of mortality; the role of this multiplication in the pathogenesis was not determined. It is proposed that toxic material is released when SSII-1 cells are digested and that multiplication of B. sphaericus in the larval gut is not essential in the pathogenesis. There appears to be no difference in the pathogenesis when differing numbers of B. sphaericus. i.e., LD_10–20 or LD_70–98 dosages, are ingested. Possible nature of the toxic material is discussed.