Susceptibility of larvae of Trichoplusia ni and Anticarsia gemmatalis to intrahemocoelic injections of conidia and blastospores of Nomuraea rileyi

The LD₅₀ for larvae of Trichoplusia ni injected with blastospores of Nomuraea rileyi was 4.30 ± 1.16 hyphal bodies/larva; the LD₅₀ for injected conidia was ca. 25,000 conidia/larva. The dose-mortality regression line for blastospores was Y = 4.6504 + 0.5487 X. Larval mortalities of Anticarsia gemmatalis and T. ni at 100 blastospores/larva were 0.4 ± 0.5% and 96.7 ± 1.9%, respectively. At a dosage of 25,000 conidia/larva, larval mortalities for A. gemmatalis and T. ni were 0.4 ± 0.5% and 43.1 ± 8.7%, respectively. Thus, larvae of A. gemmatalis were > 100 times and >200 times more resistant to injected conidia and blastospores, respectively, than were larvae of T. ni. Resistance of A. gemmatalis to N. rileyi may not be solely at the integumental barrier, as is often believed, but may also be a function of an internal physiological response.     

Biochemical characterization of digestive membrane‐associated alkaline phosphatase from the velvet bean caterpillar Anticarsia gemmatalis

In Brazil, the use of transgenic plants expressing the insect‐toxic Bacillus thuringiensis endotoxin has been successfully used as pest control management since 2013 in transgenic soybean lineages against pest caterpillars such as Helicoverpa armigera. These toxins, endogenously expressed by the plants or sprayed over the crops, are ingested by the insect and bind to receptors in the midgut of these animals, resulting in disruption of digestion and lower insect survival rates. Here, we identified and characterized a membrane‐associated alkaline phosphatase (ALP) in the midgut of Anticarsia gemmatalis, the main soybean defoliator pest in Brazil, and data suggested that it binds to Cry1Ac toxin in vitro. Our data showed a peak of ALP activity in homogenate samples of the midgut dissected from the 4th and 5th instars larvae. The brush border membrane vesicles obtained from the midgut of these larvae were used to purify a 60 kDa ALP, as detected by in‐gel activity and in vitro biochemical characterization using pharmacological inhibitors and mass spectrometry. When Cry1Ac toxin was supplied to the diet, it was efficient in decreasing larval weight gain and survival. Indeed, in vitro incubation of Cry1Ac toxin with the purified ALP resulted in a 43% decrease in ALP specific activity and enzyme‐linked immunosorbent assay showed that ALP interacts with Cry1Ac toxin in vitro, thus suggesting that ALP could function as a Cry toxin ligand. This is a first report characterizing an ALP in A. gemmatalis.     

Comparative susceptibility of two different hosts to genotypic variants of the Anticarsia gemmatalis nuclear polyhedrosis virus

The susceptibility of third instar larvae of Anticarsia gemmatalis (Lepidoptera: Noctuidae) and Diatraea saccharalis (Lepidoptera: Pyralidae) to ten distinct plaque purified genotypic variants of a selected isolate of the Anticarsia gemmatalis multiple-embedded nuclear polyhedrosis virus (AgMNPV), was compared. Despite the fact that this isolate, AgMNPV-Ds20, represents a wild strain of the AgMNPV selected for higher virulence to D. saccharalis, an alternate host, most of the variants are much more virulent to the original host Anticarsia than to Diatraea. Bioassays have shown an over one hundred-fold variation in LD₅₀ values ranging from 1700 polyhedron inclusion bodies (PIBs) to more than 200 000 PIBs/larva. The PIB production in infected larvae increased with the pathogenicity of the variant to the host, showing an average ten-fold reduction in Diatraea when compared to Anticarsia for the same variant. The virus particle yield ranged from 6×10⁷ to more than 10⁹ PIBs/g of infected larvae in Diatraea and from 8×10⁸ to more than 10¹⁰ PIBs/g of infected Anticarsia larvae. The data show a clear difference of the pathogenicity of the genotypic variants of AgMNPV in vivo both between the original and alternate host and between the individual variants for the same host. These differences found in vivo indicate that monitoring of shifts in variant frequency of wild and laboratory-propagated viral isolates in these highly heterogeneous populations would help ensure the efficacy of biological control programs.     

Lipid composition of the entomopathogenic fungus Nomuraea rileyi

The total lipid content, neutral and polar lipid composition, and fatty acid profiles were determined for the different developmental stages of the entomopathogenic fungus, Nomuraea rileyi. In general, the lipid composition of N. rileyi was found to be very similar to that of other Fungi Imperfecti. Triacylglycerides and sterols were the major neutral lipids and phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine were the major polar lipids. Phosphatidylcholine, resolved in conidial and mycelial samples, was not detected in hyphal body samples. The major fatty acids were palmitic, oleic, and linoleic. Stearic acid was detected as a minor component of hyphal body samples. During conidiogenesis the overall lipid composition was altered in (1) a reduction in total lipid content, (2) an increase in the polar/neutral lipid ratio, and (3) an increase in the degree of fatty acid unsaturation.     

Cytochrome P-450-dependent monooxygenase activity in the velvetbean caterpillar, Anticarsia gemmatalis Hübner

The activity of microsomal aldrin epoxidase was observed in various tissues of the last instar larva of the velvetbean caterpillar (Anticarsia gemmatalis Hübner) with the midgut being the most active. Optimum conditions for assaying the enzyme were established. Allelochemicals (monoterpenes, indoles and flavones) and host plants (cowpeas, cotton, peanuts, soybeans and hairy indigo) were found to induce the monooxygenase. Piperonyl butoxide decreased and increased the toxicity of methomyl and methyl parathion, respectively, suggesting that the monooxygenase system of velvetbean caterpillars plays a major role in the detoxification of xenobiotics.     

Growth characteristics of a continuous cell line from the velvetbean caterpillar,Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae)

Summary The cell line UFL-AG-286 was established from the embryos ofAnticarsia gemmatalis (Lepidoptera: Noctuidae). The cell line was characterized by isozyme analysis and from molecular weight determination of the restriction endonuclease bands of the mitochondrial DNA. Cells seeded in 24-cluster wells had a doubling time of 5.9 d when seeded at 2×10⁵ cells ml and 6.7 d when seeded at 3×10⁵ cells/ml. This work was funded by U.S. Department of Agriculture grant 84-CRCR-1-1431. Florida Agricultural Experiment Station Journal Series, no. 8181.     

Density‐dependent prophylaxis in primary anti‐parasite barriers in the velvetbean caterpillar

1. Organisms rely on a set of primary barriers to prevent invasion by parasites, and secondary defences to fight parasites that breach the primary barriers. However, maintaining these defences to be active and effective is costly. Thus, hosts increase investment in anti‐parasite defences under situations of high risk of infection and reduce defences when the risk is reduced (the ‘Density‐Dependent Prophylaxis’ hypothesis). 2. In the present study, it was tested whether the midgut primary defences of the velvetbean caterpillar Anticarsia gemmatalis Hübner present density‐dependent plasticity, and also whether these defences could be induced by a viral pathogenic challenge. The aim was to examine whether morphometry and the structure of the midgut and peritrophic matrix (PM) change in accordance with colour transition in caterpillars, and whether such changes may provide the caterpillars a more protective barrier against invasion by Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV). 3. It was found that PM and the midgut epithelium of the velvetbean caterpillar change plastically according to phenotype, itself a response to changes in population density. Caterpillars reared at high densities (black phenotype) had a considerably thicker midgut epithelia and peritrophic matrices than those reared individually (green phenotype), and there was also more chitin in the PM of the former. 4. This was interpreted as the first demonstration of increased investment in primary, barrier, defences against parasites, in response to increased conspecific density and an increased risk of infection. The possibility that this arises as a positive result of pleiotropy is discussed further, wherein the biochemical pathways responsible for the up‐regulation of the immune system are also involved in midgut properties.     

Food consumption and utilization responses to dietary dilution with cellulose and water by velvetbean caterpillars, Anticarsia gemmatalis

Abstract Dilution of an artificial diet with water or cellulose to nutrient levels of 32% (undiluted), 19% and 10% fresh weight (fw) resulted in increased fw and dry weight (dw) food consumption (both absolute amounts and weight-relative rates) by velvetbean caterpillars, Anticarsia gemmatalis Hübner. Despite these increases, the absolute amount and relative rate of nutrient intake by the caterpillars declined with dilution, as did their dw gain and dw (% fw) and lipid (% dw) contents. The proportion of consumed food (dw, including cellulose) that was digested and absorbed declined with increased dietary cellulose; however, the proportion of consumed nutrients (dw, excluding cellulose) that was digested and absorbed was not affected by the presence of cellulose, although it increased slightly (but significantly) with dietary water. The efficiency with which the absorbed nutrients were converted to biomass energy showed a negative relationship with the various measures of food consumption and thus a positive relationship with dietary nutrient level.
The compensatory increases in feeding mitigated the deleterious impact of dietary dilution on growth, which would have declined further without the increased feeding. These data suggest that food consumption is regulated primarily through an evaluation of food nutrient level, and that the metabolic costs associated with the increased consumption and processing of food may be substantial, especially on the most diluted diets. Another hypothesized cost of increased feeding, reduced efficiency of digestion and absorption as food presumably passes more rapidly through the gut, was not detected.     

A temperature-dependent developmental model for a nucleopolyhedrosis virus of the velvetbean caterpillar,Anticarsia gemmatalis (Lepidoptera: Noctuidae)

Developmental times and rates of nucleopolyhedrosis virus infection in third-instar velvetbean caterpillar, Anticarsia gemmatalis, larvae were studied in the laboratory at a variety of constant and variable temperatures. Developmental time was considered to be time from inoculation until death. Viral infection exhibited a temperature optimum of ca. 30°C and was inhibited at 10 and 40°C. Mean developmental time of the virus ranged from 18.1 days (15°C) to 5.5 days (30°C). Means and standard deviations of viral developmental rates (= development time^?1) were used as inputs into a previously derived absolute reaction rate model designed to generate a set of kinetic constants usable in predicting developmental times. Actual distributions of viral cohort developmental times were compared to distributions generated by the model. Reasonable agreement between predicted and actual distributions was found at three of four temperatures tested.     

Predatory behaviour of Podisus nigrispinus (Heteroptera: Pentatomidae) on different densities of Anticarsia gemmatalis (Lepidoptera: Noctuidae) larvae

The functional response of the predatory bug Podisus nigrispinus (Dallas) (Heteroptera: Pentatomidae) feeding on its prey, Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae), was studied in a greenhouse compartment. Each cage enclosed three soybean plants plus two, four, six, eight, 10, 12 or 14 prey larvae. One adult predator was released and kept inside the cages for 24h. The predation rate of adult male P. nigrispinus was highest at densities of eight or more A. gemmatalis larvae with a handling time of 5.76h and an attack rate of 0.68h^?1. Adult females had higher predation rates on plants with 10 or more A. gemmatalis larvae, with a handling time of 3.84h and an attack rate of 0.65h^?1. The maximum number of larvae consumed by males and females of this predator were 4.1 and 6.0 per day, respectively, in groups of three plants. The results suggest that P. nigrispinus may be used in biological control programmes against A. gemmatalis in soybean fields.     

Simultaneous monitoring of flight and oviposition of individual velvetbean caterpillar moths (by Wales, Barfield & Leppla, 1985): a critique

ABSTRACT. Flight durations of tethered female Anticarsia gemmatalis moths recorded in the laboratory were interpreted by Wales et al. as indicating a capacity for significant inter-reproductive displacements in the field. Indications that their data may not support this conclusion are discussed and evidence is presented that, in tethered Spodoptera exempta , prolonged flights starting early in the night represent migratory behaviour while those of similar durations starting after midnight do not.     

Effects of successivein vitro andIn vivo passages on the virulence of the entomopathogenic fungus,Nomuraea rileyi

There was no significant decrease or increase in the activity of conidia ofNomuraea rileyi after 12_{in vivo} serial passages in larvae ofTrichoplusia ni (Hübner) or after 12in vitro serial passages on a semi-synthetic medium. The LC-50 at the start of the serial passage was 16.8±4.0 conidia/mm²; after 12 serial passages the LC-50 for thein vitro- andin vivo-produced conidia was 16.2±2.5 conidia/mm² and 12.0±1.9 conidia/mm², respectively.

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Résumé Il n'y a pas d'augmentation ou de réduction significative dans l'activité des conidies deNomuraea rileyi après 12 passages en sériein vivo chez les larves deTrichoplusia ni (Hübner) ou après 12 passages en sériein vitro sur un milieu semi-synthétique. La concentration léthale 50 au début de ces passages était de 16,8±4,0 conidies/mm², après 12 passages la CL 50 des conidies produitesin vitro etin vivo fut respectivement de 16,2±2,5 conidies/mm² et de 12,0±1,9 conidies/mm².

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Mention of a proprietary product in this paper does not constitute a recommendation for use by the USDA.     

Cuticular and non-cuticular substrate influence on expression of cuticle-degrading enzymes from conidia of an entomopathogenic fungus,Nomuraea rileyi

Larval cuticle fromTrichoplusia ni, Helicoverpa (=Heliothis)zea, andHeliothis virescens and a cellulose substrate were used to quantify release of proteolytic, chitinolytic, and lipolytic enzymes by germinating conidia of the entomopathogenic fungus,Nomuraea rileyi. There was no significant difference in conidial viability incubated withT. ni, H. zea or cellulose substrates. Conidial viability onH. virescens cuticle, however, was significantly lower (ca. 19–25%) than the other three substrates. The presence of cuticle substrates, especially cuticle ofT. ni, stimulated germination. The nature of the substrate influenced both the time and quantity of the enzymes expressed. Specific proteases (aminopeptidase, chymoelastase, trypsin) generally were expressed earlier and/or in greater quantities on cuticular than on the cellulose substrate. Although both chitinolytic enzymes (endochitinase, N-acetylglucosaminidase) were detected on all three cuticular substrates, their activity was substantially lower than that of the proteolytic enzymes. Lipase activity was only minimally present. Early concurrent release of both proteases and chitinases suggested that both may be important in the penetration of the larval integument by germinating conidia ofN. rileyi. Expression of proteases and chitinases, especially aminopeptidase and endochitinase was probably a specific response to cuticle, because little or no activity was expressed on the non-host, cellulose substrate.This article reports the results of research only. Mention of a proprietary product in this paper does not constitute a recommendation for use by the US Department of Agriculture.     

Ultrastructural studies of spermatogenesis in the anthocerotales. III. Gamete morphogenesis: From spermatogenous cell through midstage spermatid

An ultrastructural examination of spermatogenesis in Phaeoceros has shown nucleoli to be present in spermatogenous cells and to persist until the centrioles become associated with nuclei of young spermatids. At the onset of multilayered structure (MLS) formation, well-defined aggregations of osmiophilic strands begin to form in the nuclei of young spermatids and disappear shortly after chromatin condensation starts in the midstage spermatids. When the centrioles in the young spermatids are orientated perpendicular to the nuclear envelope, the nucleoplasm immediately in front of them is densely stained. Where the spline tubules of the MLS extend over the nucleus, the nuclear envelope is devoid of pores, and the inner nuclear membrane is contacted internally by the local deposition of dense staining nucleoplasm. Chromatin condensation begins with strands extending perpendicularly from the dense staining nucleoplasm beneath the spline and continues with the nuclear beak becoming filled with condensed chromatin. As the MLS lamellae disappear acropetally, the rear portion of the anterior mitochondrion (AM) extends back under the nuclear beak which now narrows to a size that approximates the anterior end of the nucleus of a spermatozoid. By the end of the mid-spermatid stage, the nucleus has coiled approximately one gyre of a helix and the five or six central slpine tubules extend over the plastid which is now located beneath the front end of the AM. Several profiles of endoplasmic reticulum confluent with the nuclear envelope are present. Possible factors which might play a role in determining the morphology of the mid-spermatids are discussed.     

Effects of environmental factors on virulence of the entomopathogenic fungus, Nomuraea rileyi, against the corn earworm, Helicoverpa armigera (Lep., Noctuidae)

The effects of environmental factors on infection of the entomopathogenic fungus, Nomuraea rileyi , isolated from the corn earworm, Helicoverpa armigera , in Taiwan, to its host insect were studied in the laboratory. The fungus caused higher larval mortality at 20°C than at 30°C when 5 × 10⁶ conidia/ml were sprayed on the fourth instar. However, mortality of the fifth instar injected with 1 × 10³ conidia/larva was not significantly different when the inoculated larvae were incubated from 15 to 30°C. The fungal development in inoculated larvae was best at 20 and 25°C after shifting from 20°C to either lower or higher temperatures. The germination rate was higher at 20 and 25°C than at 30 or 35°C. Conidial germination was better on the wash-off of insect cuticle than on Sabouraud maltose agar with yeast extract. Sporulation on chill-dried cadavers was maximal at 95 or 100% relative humidity than at lower levels of relative humidity. The time required for sporulation was 2 days less at 100% than at 95% relative humidity. Although photoperiod did not affect fifth instar mortality caused by N. rileyi , the median lethal time (LT₅₀) values were shorter upon incubating under light than in darkness. Incubation of infected cadavers under 12 or 24 h light resulted in 20-fold more conidial production than under full darkness. Therefore, illumination is necessary for development of this isolate on insect cadavers.     

Epizootics of the entomopathogenic fungus,Entomophaga grylli (Entomophthorales: Entomophthoraceae), in a grasshopper population in Northwest China

Plagues caused by locusts and grasshoppers have led to severe crop damage and great economic loss in many countries. Nevertheless, populations of these pests are often suppressed by naturally occurring predators and disease. Among such natural control factors, the entomopathogenic fungus, Entomophaga grylli, can markedly disrupt the dynamics of grasshopper populations. However, there are few reports of epizootics of this entomopathogenic fungus occurring in consecutive years. Here we report on a consecutive 2-year field survey of E. grylli epizootics in Xinjiang, Northwest China. E. grylli were observed to infect at least four species of grasshopper, Calliptamus italicus, Gomphocerus sibiricus, Chorthippus sp., and Stauroderus scalaris. This is the first record of infection of the last two species by this pathogen. The highest infection rates at the two study sites (Chonghuer and Jiadengyu, Altay Prefecture) were ≥50%, observed in mid-July 2011, and the lowest rate was >16% in early summer. The density of infected grasshoppers was positively correlated with the density of total grasshoppers collected (r?=?0.981). Therefore, E. grylli is an important natural factor regulating the dynamics of grasshopper populations in regions that are not subjected to artificial treatments.     

Effect of the interaction between Anticarsia gemmatalis multiple nucleopolyhedrovirus and Epinotia aporema granulovirus,on A. gemmatalis (Lepidoptera: Noctuidae) larvae

The bean shoot borer Epinotia aporema Wals. (Lepidoptera: Tortricidae) and the velvet bean caterpillar Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae) are key pests of soybean and other legume crops in South America. They are often found simultaneously in certain regions. A. gemmatalis nucleopolyhedrovirus (AgMNPV) is widely used to control A. gemmatalis. More recently, E. aporema granulovirus (EpapGV) has been characterized and evaluated as a bioinsecticide for E. aporema. In order to increase its potential use and to design optimized strategies for the management of lepidopteran pests, we evaluated the interaction between EpapGV and AgMNPV on third instar A. gemmatalis larvae. Larvae fed with 50 AgMNPV OBs/larva showed an increase in the mortality rates (from 42% to 81%) and a decrease in the median survival time (from 7.7 days to 5.7 days) when these OBs were mixed with 6000 EpapGV OBs/larva. When 300 AgMNPV OBs/larva were used alone or in combination with EpapGV OBs no changes in biological parameters were observed. No mortality was detected in A. gemmatalis larvae treated with EpapGV alone. In larvae fed with the viral mixtures, only AgMNPV DNA was detected by PCR. A. gemmatalis peritrophic membranes (PMs) examined by SDS–PAGE and scanning electron microscopy showed signs of damage. Notably, we found the presence of spheroidal bodies associated with damaged areas in the PMs of larvae fed with EpapGV but not in those that were given AgMNPV alone. These results show that EpapGV increases the viral potency of AgMNPV, and thus the insecticidal efficiency, suggesting that the use of formulations including both viruses might be a valuable tool for pest management.     

落叶松-杨栅锈菌在感病杨树寄主上发育过程的组织学研究

采用荧光染色技术、光学显微镜和电子显微镜技术,系统研究了落叶松-杨栅锈菌在感病杨树叶片上的发育过程。结果表明,在侵染前期（接种12h以内）,锈菌夏孢子在杨树叶片上萌发,利用芽管或附着胞穿透叶表气孔后形成气孔下囊,进而在胞间产生侵染菌丝。进入活体营养生长阶段（接种后24-96h）,锈菌不断产生大量吸器来满足营养需求的同时,侵染菌丝在叶肉细胞间隙蔓延分枝生长至形成菌落结构。最终在产孢阶段（接种120h之后）产孢菌丝分化形成的夏孢子在表皮下聚集成堆,待成熟后突破表皮显露出来。     

Ultrastructural studies on the microfilaria of Cardianema sp. Alicata, 1933 (Nematoda; Onchocercidae)

The ultrastructure of the sheath, cuticle and hypodermis of the microfilaria of Cardianema sp, is described from electron micrographs of in utero- and blood-stages. The trilaminiar sheath invests the microfilaria throughout development in utero and it acquires a superficial coat after the microfilaria enters the blood stream of its reptile host. The cuticle consists of external and internal cortex, fibrillar and subfibrillar layers. The cuticle is attached to the hypodermis without the intervention of a basal lamina. The structure of the external cortex is modified in the annular furrows in the cuticle. The cellular hypodermis forms a complete subcuticular layer, although over much of the circumference the cells exist as thin cytoplasmic processes and where these overlap there are extensive tight junctions. The case for classifying the microfilaria of Cardianema a first stage larva is advanced and a functional but speculative, role for the sheath is proposed.