Insecticidal Potential of Defense Metabolites from Ocimum kilimandscharicum against Helicoverpa armigera

Genus Ocimum contains a reservoir of diverse secondary metabolites, which are known for their defense and medicinal value. However, the defense-related metabolites from this genus have not been studied in depth. To gain deeper insight into inducible defense metabolites, we examined the overall biochemical and metabolic changes in Ocimum kilimandscharicum that occurred in response to the feeding of Helicoverpa armigera larvae. Metabolic analysis revealed that the primary and secondary metabolism of local and systemic tissues in O. kilimandscharicum was severely affected following larval infestation. Moreover, levels of specific secondary metabolites like camphor, limonene and β-caryophyllene (known to be involved in defense) significantly increased in leaves upon insect attack. Choice assays conducted by exposing H. armigera larvae on O. kilimandscharicum and tomato leaves, demonstrated that O. kilimandscharicum significantly deters larval feeding. Further, when larvae were fed on O. kilimandscharicum leaves, average body weight decreased and mortality of the larvae increased. Larvae fed on artificial diet supplemented with O. kilimandscharicum leaf extract, camphor, limonene and β-caryophyllene showed growth retardation, increased mortality rates and pupal deformities. Digestive enzymes of H. armigera - namely, amylase, protease and lipase- showed variable patterns after feeding on O. kilimandscharicum, which implies striving of the larvae to attain required nutrition for growth, development and metamorphosis. Evidently, selected metabolites from O. kilimandscharicum possess significant insecticidal activity.     

Impact of Ocean Warming and Ocean Acidification on Larval Development and Calcification in the Sea Urchin Tripneustes gratilla

Background

As the oceans simultaneously warm, acidify and increase in P _CO2, prospects for marine biota are of concern. Calcifying species may find it difficult to produce their skeleton because ocean acidification decreases calcium carbonate saturation and accompanying hypercapnia suppresses metabolism. However, this may be buffered by enhanced growth and metabolism due to warming.

Methodology/Principal Findings

We examined the interactive effects of near-future ocean warming and increased acidification/P _CO2 on larval development in the tropical sea urchin Tripneustes gratilla. Larvae were reared in multifactorial experiments in flow-through conditions in all combinations of three temperature and three pH/P _CO2 treatments. Experiments were placed in the setting of projected near future conditions for SE Australia, a global change hot spot. Increased acidity/P _CO2 and decreased carbonate mineral saturation significantly reduced larval growth resulting in decreased skeletal length. Increased temperature (+3°C) stimulated growth, producing significantly bigger larvae across all pH/P _CO2 treatments up to a thermal threshold (+6°C). Increased acidity (-0.3-0.5 pH units) and hypercapnia significantly reduced larval calcification. A +3°C warming diminished the negative effects of acidification and hypercapnia on larval growth.

Conclusions and Significance

This study of the effects of ocean warming and CO₂ driven acidification on development and calcification of marine invertebrate larvae reared in experimental conditions from the outset of development (fertilization) shows the positive and negative effects of these stressors. In simultaneous exposure to stressors the dwarfing effects of acidification were dominant. Reduction in size of sea urchin larvae in a high P _CO2 ocean would likely impair their performance with negative consequent effects for benthic adult populations.     

Larval Behaviours and Their Contribution to the Distribution of the Intertidal Coral Reef Sponge Carteriospongia foliascens

Sponges (Phylum Porifera) are an evolutionary and ecologically significant group; however information on processes influencing sponge population distributions is surprisingly limited. Carteriospongia foliascens is a common Indo-Pacific sponge, which has been reported from the intertidal to the mesophotic. Interestingly, the distribution of C. foliascens at inshore reefs of the Great Barrier Reef is restricted to the intertidal with no individuals evident in adjacent subtidal habitats. The abundance of C. foliascens and substrate availability was first quantified to investigate the influence of substrate limitation on adult distribution. Pre-settlement processes of larval spawning, swimming speeds, phototaxis, vertical migration, and settlement to intertidal and subtidal substrate cues were also quantified. Notably, suitable settlement substrate (coral rubble) was not limiting in subtidal habitats. C. foliascens released up to 765 brooded larvae sponge⁻¹ day⁻¹ during the day, with larvae (80%±5.77) being negatively phototactic and migrating to the bottom within 40 minutes from release. Subsequently, larvae (up to 58.67%±2.91) migrated to the surface after the loss of the daylight cue (nightfall), and after 34 h post-release >98.67% (±0.67) of larvae had adopted a benthic habit regardless of light conditions. Intertidal and subtidal biofilms initiated similar settlement responses, inducing faster (as early 6 h post-release) and more successful metamorphosis (>60%) than unconditioned surfaces. C. foliascens has a high larval supply and larval behaviours that support recruitment to the subtidal. The absence of C. foliascens in subtidal habitats at inshore reefs is therefore proposed to be a potential consequence of post-settlement mortalities.     

Hermetia illucens L. larvae–associated intestinal microbes reduce the transmission risk of zoonotic pathogens in pig manure

Black soldier fly (BSF) larvae are considered a promising biological reactor to convert organic waste and reduce the impact of zoonotic pathogens on the environment. We analysed the effects of BSF larvae on Staphylococcus aureus and Salmonella spp. populations in pig manure (PM), which showed that BSF larvae can significantly reduce the counts of the associated S. aureus and Salmonella spp. Then, using a sterile BSF larval system, we validated the function of BSF larval intestinal microbiota in vivo to suppress pathogens, and lastly, we isolated eight bacterial strains from the BSF larval gut that inhibit S. aureus. Results indicated that functional microbes are essential for BSF larvae to antagonise S. aureus. Moreover, the analysis results of the relationship between the intestinal microbiota and S. aureus and Salmonella spp. showed that Myroides, Tissierella, Oblitimonas, Paenalcalignes, Terrisporobacter, Clostridium, Fastidiosipila, Pseudomonas, Ignatzschineria, Savagea, Moheibacter and Sphingobacterium were negatively correlated with S. aureus and Salmonella. Overall, these results suggested that the potential ability of BSF larvae to inhibit S. aureus and Salmonella spp. present in PM is accomplished primarily by gut‐associated microorganisms.

Black soldier fly larvae can inhibit zoonotic pathogens in pig manure. Intestinal microbes are essential for black soldier fly larvae to antagonise zoonotic pathogens. Black soldier fly larvae and their intestinal microbes can reduce zoonotic pathogens transmission risk in pig manure.     

Aspects of Embryonic and Larval Development in Bighead Carp Hypophthalmichthys nobilis and Silver Carp Hypophthalmichthys molitrix

As bighead carp Hypophthalmichthys nobilis and silver carp H . molitrix (the bigheaded carps) are poised to enter the Laurentian Great Lakes and potentially damage the region’s economically important fishery, information on developmental rates and behaviors of carps is critical to assessing their ability to establish sustainable populations within the Great Lakes basin. In laboratory experiments, the embryonic and larval developmental rates, size, and behaviors of bigheaded carp were tracked at two temperature treatments, one “cold” and one “warm”. Developmental rates were computed using previously described stages of development and the cumulative thermal unit method. Both species have similar thermal requirements, with a minimum developmental temperature for embryonic stages of 12.1° C for silver carp and 12.9° C for bighead carp, and 13.3° C for silver carp larval stages and 13.4° C for bighead carp larval stages. Egg size differed among species and temperature treatments, as egg size was larger in bighead carp, and “warm" temperature treatments. The larvae started robust upwards vertical swimming immediately after hatching, interspersed with intervals of sinking. Vertical swimming tubes were used to measure water column distribution, and ascent and descent rates of vertically swimming fish. Water column distribution and ascent and descent rates changed with ontogeny. Water column distribution also showed some diel periodicity. Developmental rates, size, and behaviors contribute to the drift distance needed to fulfill the early life history requirements of bigheaded carps and can be used in conjunction with transport information to assess invasibility of a river.     

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism Drosophila melanogaster, inductive microenvironments known as larval Hematopoietic Pockets (HPs) have been identified as anatomical sites for the development and regulation of blood cells (hemocytes), in particular of the self-renewing macrophage lineage. HPs are segmentally repeated pockets between the epidermis and muscle layers of the larva, which also comprise sensory neurons of the peripheral nervous system. In the larva, resident (sessile) hemocytes are exposed to anti-apoptotic, adhesive and proliferative cues from these sensory neurons and potentially other components of the HPs, such as the lining muscle and epithelial layers. During normal development, gradual release of resident hemocytes from the HPs fuels the population of circulating hemocytes, which culminates in the release of most of the resident hemocytes at the beginning of metamorphosis. Immune assaults, physical injury or mechanical disturbance trigger the premature release of resident hemocytes into circulation. The switch of larval hemocytes between resident locations and circulation raises the need for a common standard/procedure to selectively isolate and quantify these two populations of blood cells from single Drosophila larvae. Accordingly, this protocol describes an automated method to release and quantify the resident and circulating hemocytes from single larvae. The method facilitates ex vivo approaches, and may be adapted to serve a variety of developmental stages of Drosophila and other invertebrate organisms.     

A Glycoprotein in Shells of Conspecifics Induces Larval Settlement of the Pacific Oyster Crassostrea gigas

Settlement of larvae of Crassostrea gigas on shell chips (SC) prepared from shells of 11 different species of mollusks was investigated. Furthermore, the settlement inducing compound in the shell of C. gigas was extracted and subjected to various treatments to characterize the chemical cue. C. gigas larvae settled on SC of all species tested except on Patinopecten yessoensis and Atrina pinnata. In SC of species that induced C. gigas larvae to settle, settlement was proportionate to the amount of SC supplied to the larvae. When compared to C. gigas SC, all species except Crassostrea nippona showed lower settlement inducing activities, suggesting that the cue may be more abundant or in a more available form to the larvae in shells of conspecific and C. nippona than in other species. The settlement inducing activity of C. gigas SC remained intact after antibiotic treatment. Extraction of C. gigas SC with diethyl ether (Et₂O-ex), ethanol (EtOH-ex), and water (Aq-ex) did not induce larval settlement of C. gigas larvae. However, extraction of C. gigas SC with 2N of hydrochloric acid (HCl-ex) induced larval settlement that was at the same level as the SC. The settlement inducing compound in the HCl-ex was stable at 100°C but was destroyed or degraded after pepsin, trypsin, PNGase F and trifluoromethanesulfonic acid treatments. This chemical cue eluted between the molecular mass range of 45 and 150 kDa after gel filtration and revealed a major band at 55 kDa on the SDS-PAGE gel after staining with Stains-all. Thus, a 55 kDa glycoprotein component in the organic matrix of C. gigas shells is hypothesized to be the chemical basis of larval settlement on conspecifics.     

Embryogenesis and Larval Biology of the Cold-Water Coral Lophelia pertusa

Cold-water coral reefs form spectacular and highly diverse ecosystems in the deep sea but little is known about reproduction, and virtually nothing about the larval biology in these corals. This study is based on data from two locations of the North East Atlantic and documents the first observations of embryogenesis and larval development in Lophelia pertusa, the most common framework-building cold-water scleractinian. Embryos developed in a more or less organized radial cleavage pattern from ∼160 µm large neutral or negatively buoyant eggs, to 120–270 µm long ciliated planulae. Embryogenesis was slow with cleavage occurring at intervals of 6–8 hours up to the 64-cell stage. Genetically characterized larvae were sexually derived, with maternal and paternal alleles present. Larvae were active swimmers (0.5 mm s⁻¹) initially residing in the upper part of the water column, with bottom probing behavior starting 3–5 weeks after fertilization. Nematocysts had developed by day 30, coinciding with peak bottom-probing behavior, and possibly an indication that larvae are fully competent to settle at this time. Planulae survived for eight weeks under laboratory conditions, and preliminary results indicate that these planulae are planktotrophic. The late onset of competency and larval longevity suggests a high dispersal potential. Understanding larval biology and behavior is of paramount importance for biophysical modeling of larval dispersal, which forms the basis for predictions of connectivity among populations.     

In Vivo Assessment of Cold Adaptation in Insect Larvae by Magnetic Resonance Imaging and Magnetic Resonance Spectroscopy

Background

Temperatures below the freezing point of water and the ensuing ice crystal formation pose serious challenges to cell structure and function. Consequently, species living in seasonally cold environments have evolved a multitude of strategies to reorganize their cellular architecture and metabolism, and the underlying mechanisms are crucial to our understanding of life. In multicellular organisms, and poikilotherm animals in particular, our knowledge about these processes is almost exclusively due to invasive studies, thereby limiting the range of conclusions that can be drawn about intact living systems.

Methodology

Given that non-destructive techniques like ¹H Magnetic Resonance (MR) imaging and spectroscopy have proven useful for in vivo investigations of a wide range of biological systems, we aimed at evaluating their potential to observe cold adaptations in living insect larvae. Specifically, we chose two cold-hardy insect species that frequently serve as cryobiological model systems–the freeze-avoiding gall moth Epiblema scudderiana and the freeze-tolerant gall fly Eurosta solidaginis.

Results

In vivo MR images were acquired from autumn-collected larvae at temperatures between 0°C and about −70°C and at spatial resolutions down to 27 µm. These images revealed three-dimensional (3D) larval anatomy at a level of detail currently not in reach of other in vivo techniques. Furthermore, they allowed visualization of the 3D distribution of the remaining liquid water and of the endogenous cryoprotectants at subzero temperatures, and temperature-weighted images of these distributions could be derived. Finally, individual fat body cells and their nuclei could be identified in intact frozen Eurosta larvae.

Conclusions

These findings suggest that high resolution MR techniques provide for interesting methodological options in comparative cryobiological investigations, especially in vivo.     

Edin Expression in the Fat Body Is Required in the Defense Against Parasitic Wasps in Drosophila melanogaster

The cellular immune response against parasitoid wasps in Drosophila involves the activation, mobilization, proliferation and differentiation of different blood cell types. Here, we have assessed the role of Edin (elevated during infection) in the immune response against the parasitoid wasp Leptopilina boulardi in Drosophila melanogaster larvae. The expression of edin was induced within hours after a wasp infection in larval fat bodies. Using tissue-specific RNAi, we show that Edin is an important determinant of the encapsulation response. Although edin expression in the fat body was required for the larvae to mount a normal encapsulation response, it was dispensable in hemocytes. Edin expression in the fat body was not required for lamellocyte differentiation, but it was needed for the increase in plasmatocyte numbers and for the release of sessile hemocytes into the hemolymph. We conclude that edin expression in the fat body affects the outcome of a wasp infection by regulating the increase of plasmatocyte numbers and the mobilization of sessile hemocytes in Drosophila larvae.     

Characterisation of Culex quinquefasciatus (Diptera: Culicidae) larval habitats at ground level and temporal fluctuations of larval abundance in Córdoba, Argentina

The aims of this study were to characterise the ground-level larval habitats of the mosquito Culex quinquefasciatus, to determine the relationships between habitat characteristics and larval abundance and to examine seasonal larval-stage variations in Córdoba city. Every two weeks for two years, 15 larval habitats (natural and artificial water bodies, including shallow wells, drains, retention ponds, canals and ditches) were visited and sampled for larval mosquitoes. Data regarding the water depth, temperature and pH, permanence, the presence of aquatic vegetation and the density of collected mosquito larvae were recorded. Data on the average air temperatures and accumulated precipitation during the 15 days prior to each sampling date were also obtained. Cx. quinquefasciatus larvae were collected throughout the study period and were generally most abundant in the summer season. Generalised linear mixed models indicated the average air temperature and presence of dicotyledonous aquatic vegetation as variables that served as important predictors of larval densities. Additionally, permanent breeding sites supported high larval densities. In Córdoba city and possibly in other highly populated cities at the same latitude with the same environmental conditions, control programs should focus on permanent larval habitats with aquatic vegetation during the early spring, when the Cx. quinquefasciatus population begins to increase.     

Soil Applications of Steinernematid and Heterorhabditid Nematodes for Control of Colorado Potato Beetles,Leptinotarsa decemlineata (Say)

Three strains of Steinernema feltiae Filipjev (All, Mexican, and Breton strains) and one of Heterorhabditis heliothidis (Khan, Brooks, and Hirschmann) were evaluated for their potential to control Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say), larvae and pupae in the soil. In laboratory studies, H. heliothidis and S. feltiae (Mexican strain) produced the highest mortality (6 days posttreatment) of CPB when applied to the surface of a soil column containing mature CPB larvae 5 cm below. Mortality ranged from 80 to 90% at rates of 79-158 nematodes/cm². Similar results were seen in a field microplot study with all four nematodes; S. feltiae (Mexican strain) and H. heliothidis were most effective. Adult CPB emergence was reduced 86.5-100% after application of 31-93 H. heliothidis/cm² and 88.4-100% with 93-155 S. feltiae (Mexican strain)/cm². The All strain of S. feltiae was moderately effective (ca. 80% reduction at 93-155 nematodes/cm²), while the Breton strain was ineffective (< 40% reduction at 155 nematodes/cm²). In small plots of potatoes enclosed in field cages, application of H. heliothidis and S. feltiae (Mexican strain) at rates of 93-155 nematodes/cm² before larval CPB burial in the soil resulted in 66-77% reduction in adult CPB emergence. Soil applications of these nematodes show potential for biological control of CPB.     

Development of the Acoustically Evoked Behavioral Response in Larval Plainfin Midshipman Fish,Porichthys notatus

The ontogeny of hearing in fishes has become a major interest among bioacoustics researchers studying fish behavior and sensory ecology. Most fish begin to detect acoustic stimuli during the larval stage which can be important for navigation, predator avoidance and settlement, however relatively little is known about the hearing capabilities of larval fishes. We characterized the acoustically evoked behavioral response (AEBR) in the plainfin midshipman fish, Porichthys notatus, and used this innate startle-like response to characterize this species'' auditory capability during larval development. Age and size of larval midshipman were highly correlated (r² = 0.92). The AEBR was first observed in larvae at 1.4 cm TL. At a size ≥1.8 cm TL, all larvae responded to a broadband stimulus of 154 dB re1 µPa or −15.2 dB re 1 g (z-axis). Lowest AEBR thresholds were 140–150 dB re 1 µPa or −33 to −23 dB re 1 g for frequencies below 225 Hz. Larval fish with size ranges of 1.9–2.4 cm TL had significantly lower best evoked frequencies than the other tested size groups. We also investigated the development of the lateral line organ and its function in mediating the AEBR. The lateral line organ is likely involved in mediating the AEBR but not necessary to evoke the startle-like response. The midshipman auditory and lateral line systems are functional during early development when the larvae are in the nest and the auditory system appears to have similar tuning characteristics throughout all life history stages.     

Patterns of Biomass and Carbon Distribution across a Chronosequence of Chinese Pine (Pinus tabulaeformis) Forests

Patterns of biomass and carbon (C) storage distribution across Chinese pine (Pinus tabulaeformis) natural secondary forests are poorly documented. The objectives of this study were to examine the biomass and C pools of the major ecosystem components in a replicated age sequence of P. tabulaeformis secondary forest stands in Northern China. Within each stand, biomass of above- and belowground tree, understory (shrub and herb), and forest floor were determined from plot-level investigation and destructive sampling. Allometric equations using the diameter at breast height (DBH) were developed to quantify plant biomass. C stocks in the tree and understory biomass, forest floor, and mineral soil (0–100 cm) were estimated by analyzing the C concentration of each component. The results showed that the tree biomass of P. tabulaeformis stands was ranged from 123.8 Mg·ha^–1 for the young stand to 344.8 Mg·ha^–1 for the mature stand. The understory biomass ranged from 1.8 Mg·ha^–1 in the middle-aged stand to 3.5 Mg·ha^–1 in the young stand. Forest floor biomass increased steady with stand age, ranging from 14.9 to 23.0 Mg·ha^–1. The highest mean C concentration across the chronosequence was found in tree branch while the lowest mean C concentration was found in forest floor. The observed C stock of the aboveground tree, shrub, forest floor, and mineral soil increased with increasing stand age, whereas the herb C stock showed a decreasing trend with a sigmoid pattern. The C stock of forest ecosystem in young, middle-aged, immature, and mature stands were 178.1, 236.3, 297.7, and 359.8 Mg C ha^–1, respectively, greater than those under similar aged P. tabulaeformis forests in China. These results are likely to be integrated into further forest management plans and generalized in other contexts to evaluate C stocks at the regional scale.     

QTL Mapping of Genome Regions Controlling Temephos Resistance in Larvae of the Mosquito Aedes aegypti

Introduction

The mosquito Aedes aegypti is the principal vector of dengue and yellow fever flaviviruses. Temephos is an organophosphate insecticide used globally to suppress Ae. aegypti larval populations but resistance has evolved in many locations.

Methodology/Principal Findings

Quantitative Trait Loci (QTL) controlling temephos survival in Ae. aegypti larvae were mapped in a pair of F₃ advanced intercross lines arising from temephos resistant parents from Solidaridad, México and temephos susceptible parents from Iquitos, Peru. Two sets of 200 F₃ larvae were exposed to a discriminating dose of temephos and then dead larvae were collected and preserved for DNA isolation every two hours up to 16 hours. Larvae surviving longer than 16 hours were considered resistant. For QTL mapping, single nucleotide polymorphisms (SNPs) were identified at 23 single copy genes and 26 microsatellite loci of known physical positions in the Ae. aegypti genome. In both reciprocal crosses, Multiple Interval Mapping identified eleven QTL associated with time until death. In the Solidaridad×Iquitos (SLD×Iq) cross twelve were associated with survival but in the reciprocal IqxSLD cross, only six QTL were survival associated. Polymorphisms at acetylcholine esterase (AchE) loci 1 and 2 were not associated with either resistance phenotype suggesting that target site insensitivity is not an organophosphate resistance mechanism in this region of México.

Conclusions/Significance

Temephos resistance is under the control of many metabolic genes of small effect and dispersed throughout the Ae. aegypti genome.     

Pharmacological properties and preliminary phytochemical analysis of Pseudocaryopteris foetida (D.Don) P.D. Cantino leaves

Medicinal plants have significant contribution in pharmaceutical industries being producers of compounds utilized as precursors for drug development. A plant of Lamiaceae family; Pseudocaryopteris foetida had not been investigated for its biomedical potential. Current research was aimed to investigate phytochemical analysis, cytotoxic potential and antioxidant activity of crude methanolic extract and fractions of Pseudocaryopteris foetida (leaves). The preliminary phytochemical analysis of crude methanolic extracts and fractions of Pseudocaryopteris foetida revealed that plant is rich in phenolic and flavonoid classes of secondary metabolites while presence of tannin was observed only in crude methanolic extract. The cytotoxicity was determined using brine shrimp lethality test. Different concentrations (25, 50, 100, 150, 200 and 250 µg/mL) of crude methanolic extract and fractions exhibited dose dependent cytotoxicity. However, The LD₅₀ for all the extracts was more than 200 µg/mL indicating weak cytotoxic potential of Pseudocaryopteris foetida. The antioxidant capabilities of crude methanolic extract and fraction of Pseudocaryopteris foetida were analyzed by in vitro bio assays including DPPH, ABTS, Reducing power and phosphomolybdate antioxidant assays using ascorbic acid as standard. The crude methanolic extract showed IC₅₀ (256.38 ± 0.6 and 314.95 ± 1.1 µg/mL) for DPPH and ABTS respectively, while total antioxidant capacity was calculated as 55.79 ± 0.5 µg/mL for crude methanolic extract of Pseudocaryopteris foetida while ascorbic acid indicated total antioxidant capacity of 71.89 ± 2.3 µg/mL. Study concluded that leaves of Pseudocaryopteris foetida were the rich source of antioxidant phytochemicals. Based on preliminary investigations further research should be focused to isolate bioactive phytochemicals as leading source of clinical medicines in future.     

Susceptibility of the Colorado Potato Beetle and the Sugarbeet Wireworm to Steinernema feltiae and S. glaseri

In laboratory tests, larvae of the Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say), and the sugarbeet wireworm (SBW), Limonius californicus (Mannerheim), were exposed to the nematodes Steinernema feltiae Filipjev (Mexican strain) (= Neoaplectana carpocapsae) and S. glaseri Steiner in soil. S. feltiae caused significantly higher mortality in SBW larvae than did S. glaseri, but both nematode species were equally effective against CPB larvae. The minimum concentration of S. feltiae for 100% mortality of CPB larvae after 13 days was 157 nematodes/cm² of soil, and the LC₅₀ based on 6-day mortality was 47.5 nematodes/cm²; in contrast, 100% mortality of SBW larvae was not achieved with even the highest concentration tested, 393 nematodes/cm². CPB adults emerging from nematode-contaminated soil were not infected. In field cage tests, S. feltiae applied to the soil surface at the rates of 155 and 310 nematodes/cm² soil caused 59% and 71% mortality, respectively, of late-fourth-instar spring-generation CPB, and 28% and 29% mortality, respectively, of SBW. No infection was obtained when larvae of summer generation CPB and SBW were placed in the same cages approximately 6 weeks after nematodes were applied to the soil. Inundative soil applications of S. feltiae, though cost prohibitive at present, were effective in reducing caged CPB and SBW field populations.     

Parallel Changes in H2O2 and Catalase during Thermotolerance Induced by Salicylic Acid or Heat Acclimation in Mustard Seedlings

Spraying mustard (Sinapis alba L.) seedlings with salicylic acid (SA) solutions between 10 and 500 μm significantly improved their tolerance to a subsequent heat shock at 55°C for 1.5 h. The effects of SA were concentration dependent, with higher concentrations failing to induce thermotolerance. The time course of thermotolerance induced by 100 μm SA was similar to that obtained with seedlings acclimated at 45°C for 1 h. We examined the hypothesis that induced thermotolerance involved H₂O₂. Heat shock at 55°C caused a significant increase in endogenous H₂O₂ and reduced catalase activity. A peak in H₂O₂ content was observed within 5 min of either SA treatment or transfer to the 45°C acclimation temperature. Between 2 and 3 h after SA treatment or heat acclimation, both H₂O₂ and catalase activity significantly decreased below control levels. The lowered H₂O₂ content and catalase activity occurred in the period of maximum thermoprotection. It is suggested that thermoprotection obtained either by spraying SA or by heat acclimation may be achieved by a common signal transduction pathway involving an early increase in H₂O₂.     

Ingestibility,Digestibility, and Engineered Biological Control Potential of Flavobacterium hibernum,Isolated from Larval Mosquito Habitats

Flavobacterium hibernum, isolated from larval habitats of the eastern tree hole mosquito, A. triseriatus, remained suspended in the larval feeding zone much longer (8 days) than other bacteria. Autofluorescent protein markers were developed for the labeling of F. hibernum with a strong flavobacterial expression system. Green fluorescent protein (GFP)-tagged F. hibernum cells were quickly consumed by larval mosquitoes at an ingestion rate of 9.5 × 10⁴/larva/h. The ingested F. hibernum cells were observed mostly in the foregut and midgut and rarely in the hindgut, suggesting that cells were digested and did not pass the gut viably. The NanoLuc luciferase reporter system was validated for quantitative larval ingestion rate and bacterial fate analyses. Larvae digested 1.87 × 10⁵ cells/larva/h, and few F. hibernum cells were excreted intact. Expression of the GFP::Cry11A fusion protein with the P20 chaperone protein from Bacillus thuringiensis H-14 was successfully achieved in F. hibernum. Whole-cell bioassays of recombinant F. hibernum exhibited high larvicidal activity against A. triseriatus in microplates and in microcosms simulating tree holes. F. hibernum cells persisted in microcosms at 100, 59, 30, and 10% of the initial densities at days 1, 2, 3, and 6, respectively, when larvae were absent, while larvae consumed nearly all of the F. hibernum cells within 3 days of their addition to microcosms.