Biocontrol potential of six Heterorhabditis bacteriophora strains isolated in the Azores Archipelago.

Entomopathogenic nematodes (EPNs) are closely associated with Popillia japonica and potentially used as their biological control agents, although field results proved inconsistent and evoked a continual pursuit of native EPNs more adapted to the environment. Therefore, we surveyed the Azorean Archipelago to isolate new strains of Heterorhabditis bacteriophora and to evaluate their virulence against the model organism Galleria mellonella under laboratory conditions. Six strains were obtained from pasture and coastal environments and both nematode and symbiont bacteria were molecularly identified. The bioassays revealed that Az172, Az186, and Az171 presented high virulence across the determination of a lethal dose (LD50) and short exposure time experiments with a comparable performance to Az29. After 72 hours, these virulent strains presented a mean determination of a lethal dose of 11 infective juveniles cm-2, a lethal time (LT50) of 34 hours, and achieved 40% mortality after an initial exposure time of only 60 minutes. Az170 exhibited an intermediate performance, whereas Az179 and Az180 were classified as low virulent strains. However, both strains presented the highest reproductive potential with means of 1700 infective juveniles/mg of larvae. The bioassays of the native EPNs obtained revealed that these strains hold the potential to be used in biological control initiatives targeting P. japonica because of their high virulence and locally adapted to environmental conditions.

Long-term suppression of turfgrass insect pests with native persistent entomopathogenic nematodes.

Entomopathogenic nematodes (EPNs) can control several important turfgrass insect pests including white grubs, weevils, cutworms, and sod webworms. But most of the research has focused on inundative releases in a biopesticide strategy using EPN strains that may have lost some of their ability to persist effectively over years of lab maintenance and / or selection for virulence and efficient mass-production. Our study examined the potential of fresh field isolate mixes of endemic EPNs to provide multi-year suppression of turfgrass insect pests. In early June 2020, we applied isolate mixes from golf courses of the EPNs Steinernema carpocapsae, Heterorhabditis bacteriophora, and their combination to plots straddling fairway and rough on two golf courses in central New Jersey, USA. Populations of EPNs and insect pests were sampled on the fairway and rough side of the plots from just before EPN application until October 2022. EPN populations increased initially in plots treated with the respective species. Steinernema carpocapsae densities stayed high for most of the experiment. Heterorhabditis bacteriophora densities decreased after 6 months and stabilized at lower levels. Several insect pests were reduced across the entire experimental period. In the fairway, the combination treatment reduced annual bluegrass weevil larvae (59 % reduction) and adults (74 %); S. carpocapsae reduced only adults (42 %). White grubs were reduced by H. bacteriophora (67 %) and the combination (63 %). Black turfgrass ataenius adults were reduced in all EPN treatments (43-62 %) in rough and fairway. Sod webworm larvae were reduced by S. carpocapsae in the fairway (75 %) and the rough (100 %) and by H. bacteriophora in the rough (75 %). Cutworm larvae were reduced in the fairway by S. carpocapsae (88 %) and the combination (75 %). Overall, our observations suggest that inoculative applications of fresh field isolate mixes of endemic EPNs may be a feasible approach to long-term suppression of insect pests in turfgrass but may require periodic reapplications.

Optimizing the releasing strategy used for the biological control of the sugarcane borer Diatraea saccharalis by Trichogramma galloi with computer modeling and simulation.

One of the challenges in augmentative biological control programs is the definition of releasing strategy for natural enemies, especially when macro-organisms are involved. Important information about the density of insects to be released and frequency of releases usually requires a great number of experiments, which implies time and space that are not always readily available. In order to provide science-based responses for these questions, computational models offer an in silico option to simulate different biocontrol agent releasing scenarios. This allows decision-makers to focus their efforts to more feasible options. The major insect pest in sugarcane crops is the sugarcane borer Diatraea saccharalis, which can be managed using the egg parasitoid Trichogramma galloi. The current strategy consists in releasing 50,000 insects per hectare for each release, in three weekly releases. Here, we present a simulation model to check whether this releasing strategy is optimal against the sugarcane borer. A sensitive analysis revealed that the population of the pest is more affected by the number of releases rather than by the density of parasitoids released. Only the number of releases demonstrated an ability to drive the population curve of the pest towards a negative growth. For example, releasing a total of 600,000 insects per hectare in three releases led to a lower pest control efficacy that releasing only 250,000 insects per hectare in five releases. A higher number of releases covers a wider range of time, increasing the likelihood of releasing parasitoids at the correct time given that the egg stage is short. Based on these results, it is suggested that, if modifications to the releasing strategy are desired, increasing the number of releases from 3 to 5 at weekly intervals is most likely preferable.

First record of Apanteles hemara (N.) on Leucinodes orbonalis Guenée and biodiversity of Hymenoptera parasitoids on Brinjal.

The brinjal fruit and shoot borer (BFSB), Leucinodes orbonalis Guenée (Lepidoptera: Crambidae), is a very detrimental pest that causes significant economic losses to brinjal crop worldwide. Infested brinjal fruits were collected from vegetable fields located at the ICAR-Indian Agricultural Research Institute (ICAR-IARI), New Delhi, India, during two consecutive seasons (2021-2022). The larvae of the pest were brought to the laboratory and reared under controlled conditions of 25 ± 0.5 °C and 70 ± 5% relative humidity, for the emergence of parasitoids. In addition, the survey of Hymenoptera parasitoids in brinjal was conducted utilizing a sweep net and yellow pan trap over the course of two seasons. The results reveal that five parasitoid species were emerged from L. orbonalis viz., Apanteles hemara Nixon, 1965, Bracon greeni Ashmead 1896 (Hymenoptera: Braconidae), Goryphus nursei (Cameron, 1907), Trathala flavoorbitalis (Cameron, 1907) (Hymenoptera: Ichneumonidae) and Spalangia gemina Boucek 1963 (Hymenoptera: Spalangiidae). Out of these, A. hemara and S. gemina were documented as new occurrences in Delhi. Additionally, A. hemara was recorded for the first time as a parasite on L. orbonalis. Trathala flavoorbitalis was observed during both seasons and exhibited higher parasitism reaching 15.55% and 18.46% in July and August 2022, respectively. However, the average parasitism (%) recorded by A. hemara, B. greeni, G. nursei, T. flavoorbitalis and S. gemina was 3.10%, 1.76%, 1.10%, 9.28% and 1.20% respectively. Furthermore, the findings showed a significant (p ≤ 0.01) strongly positive correlation between fruit infestation (%) by L. orbonalis and parasitism (%). The survey indicates the presence of a broad group (19 families and 60 species) of Hymenoptera parasitoids in the brinjal crop ecosystem in Delhi which could be valuable in biological control. In light of these results, this study revealed that A. hemara and other parasitoids identified in this study alongside T. flavoorbitalis would be ideal biocontrol agents within the integrated pest management (IPM) program of BFSB in Delhi.

Prediction of the potential distribution area of Spodoptera frugiperda and its parasitic wasp, Trichogramma pretiosum.

Spodoptera frugiperda is a migratory agricultural pest with fast-spreading speed, long migration distance, and wide host range, which seriously threatens the safety of economic crops. To predict the trends of S. frugiperda and its parasitoid wasp Trichogramma pretiosum in their habitats under current and future climatic conditions, based on MaxEnt model and geographic distribution data of their historical occurrence, we project the feasibility of introducing T. pretiosum to control S. frugiperda by evaluating on their potential global distribution. The results show that, under the current greenhouse gas concentration, the potential distribution area of S. frugiperda is concentrated in 50° N-30° S, with a total area of 1.74 × 106 km2 , and the potential distribution area of T. pretiosum in the whole world is 2.91 × 106 km2 . The suitable areas of T. pretiosum cover almost all the suitable areas of S. frugiperda, which indicates that T. pretiosum can be introduced to control S. frugiperda. The results of this study can provide a theoretical basis for the monitoring and early warning of S. frugiperda and the use of T. pretiosum to control S. frugiperda.

Effect of sublethal concentrations of the bioinsecticide spinosyn treatment of Trichoplusia ni eggs on the caterpillar and its parasitoid, Trichogramma brassicae.

BACKGROUND: Integrated Pest Management (IPM) seeks to combine multiple management strategies for optimal pest control. One method that is successfully employed in IPM is the use of beneficial organisms. However, in severe circumstances when pest insects exceed threshold limits, insecticides may still need to be implemented. Thus, understanding the effects of insecticides on biocontrol agents, such as parasitoid wasps, is paramount to ensure sustainable agroecosystems. Sublethal effects of the bioinsecticide spinosyn, a mixture of the bacterial Saccharopolyspora spinosa (Mertz and Yao) fermentation products spinosyn A and D, on eggs of Trichoplusia ni (Hübner), a cruciferous crop pest, and its egg parasitoid Trichogramma brassicae (Bezdenko) was investigated. RESULTS: The LC50 for spinosyn A and D (dissolved in ethanol) on T. ni eggs is 54 ng mL-1. Transcriptomics on caterpillars (1st and 3rd instars) that hatched from eggs treated with sublethal concentrations of spinosyn identified the upregulation of several genes encoding proteins that may be involved in insecticide resistance including detoxification enzymes, such as cytochrome P450s, glutathione S-transferases and esterases. Sublethal T. ni egg treatments did not affect parasitoid emergence, however, there was a marked increase in the size of T. brassicae hind tibia and wings that emerged from spinosyn-treated eggs. CONCLUSIONS: For the caterpillar, treatment of eggs with sublethal concentrations of spinosyn may induce insecticide resistance mechanisms. For the parasitoids, their increased size when reared in spinosyn-treated eggs suggests that the emerged wasps may have higher performance. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Improved effects of Helicoverpa armigera nucleopolyhedrovirus integrated with Campoletis chlorideae against H. armigera and impact of the virus on the parasitoid.

BACKGROUND: Combined use can be an effective measure to improve pest control by viruses and parasitic wasps. However, not all combinations of natural enemies show improved effects. Helicoverpa armigera nucleopolyhedrovirus (HearNPV) and Campoletis chlorideae Uchida are two important natural enemies of Helicoverpa armigera. This study aimed to investigate the combined effects of C. chlorideae and HearNPV against H. armigera larvae and the impact of HearNPV on C. chlorideae. RESULTS: The combination of HearNPV and C. chlorideae exerted increased mortality on H. armigera when C. chlorideae parasitized larvae one day after infection with HearNPV. C. chlorideae could distinguish between HearNPV-infected and noninfected larvae. Besides influencing host selection of C. chlorideae, HearNPV infection had negative effects on the development and reproduction of C. chlorideae. The developmental time of C. chlorideae was significantly prolonged and the percentage of emergence and adult eclosion of C. chlorideae was lower in infected hosts. The adult wasps were also smaller in body size, and female adults had fewer eggs when they developed in virus-infected hosts. CONCLUSIONS: HearNPV combined with C. chlorideae could improve the efficacy of biological control against H. armigera. The results provided valuable information on the importance of timing in the combined use of HearNPV and C. chlorideae for the biological control of H. armigera. © 2023 Society of Chemical Industry.

Relationship between parasitism and morphology of Trichogramma (Hymenoptera: Trichogrammatidae) on Spodoptera frugiperda (Lepidoptera: Noctuidae).

In this study, 5 species of Trichogramma Westwood were evaluated for the biological control of Spodoptera frugiperda (JE Smith), concerning the physical characteristics of female Trichogramma. The results showed that Trichogramma chilonis Ishii, Trichogramma dendrolimi Matsumura, and Trichogramma ostriniae Pang et Chen exhibited high parasitism rates, emergence rates, and offspring numbers, with the highest values observed for T. ostriniae. The ovipositor length of Trichogramma japonicum Ashmead and T. dendrolimi were longer than those of other species, and the hind tibia length was the shortest in Trichogramma cacoeciae Marchal. We further evaluated relationships between the parasitism ability of Trichogramma and various morphological indexes based on Spearman's rank correlation coefficients. A positive correlation was found between the parasitism rate and hind tibia length of T. cacoeciae. In T. dendrolimi, the parasitism rate was negatively correlated with ovipositor width and positively correlated with the length-width ratio of the ovipositor. A significant positive correlation was observed between the proportion of female offspring and the mother's ovipositor length in T. japonicum. However, there were no significant correlations between morphological indexes and indexes of parasitism in T. ostriniae. Overall, the parasitic abilities of T. chilonis on S. frugiperda eggs were significantly correlated with the morphology of the female ovipositors.

Dynamics of the natural enemy community of Hyphantria cunea (Lepidoptera: Erebidae) in Dandong, China.

This study aims to explore the composition of natural enemy species in the fall webworm, Hyphantria cunea (Drury) population and the dynamics of its natural enemy community in Dandong, Liaoning Province, China, where it was first reported. We collected the natural enemy of eggs, larvae, and pupae of H. cunea on host trees at 12 survey sites from June 2019 to October 2020. The results showed that the community consists of 34 species: 20 predatory species, including 15 spiders and 5 insects, and 14 parasitic species, including 10 parasitic wasps and 4 parasitic flies. The top 3 dominant species based on the importance value index for both parasitic and predatory species were Pediobius pupariae > Chouioia cunea > Cotesia gregalis in the natural enemy community of H. cunea. Analysis of all 3 principal components by principal component analysis showed that Clubionidae sp. 1, Parena cavipennis, or other predators were the main factors affecting the natural enemy community. Analysis of the community structure parameters of the H. cunea natural enemy community in different developmental stages across generations revealed the following: (i) Compared with the degree of complexity of the egg and pupal stages, the larval stage was the highest. (ii) The complexity was determined by means of comprehensive evaluation: first-generation larvae in 2020 > first-generation larvae in 2019 > second-generation larvae in 2020 > second-generation larvae in 2019. These results clarify the dynamics of natural enemy species, coevolution with the host in the invaded habitat of H. cunea and development of biological control technologies.

Involvement of a novel Pseudomonas protegens strain associated with entomopathogenic nematode infective juveniles in insect pathogenesis.

BACKGROUND: The bioinsecticidal action of entomopathogenic nematodes (EPNs) typically relies on their symbiosis with core bacteria. However, recent studies highlighted the possible involvement of other noncore species. We have recently isolated a novel Pseudomonas protegens strain as a major agent of septicaemia in larvae of the wax moth, Galleria mellonella, infected with a soil-dwelling Steinernema feltiae strain. The actual role of this bacterium in entomopathogenesis was investigated. RESULTS: The association of P. protegens with nematodes appeared to be robust, as supported by its direct and repeated isolation from both nematodes and insect larvae infected for several consecutive generations. The bacterium appeared to be well-adapted to the insect haemocoel, being able to proliferate rapidly after the injection of even a small amount of living cells [100 colony forming units (CFU)] to a larva, causing its fast death. The bacterium also was able to act by ingestion against G. mellonella larvae [median lethal concentration (LC50 ) = 4.0 × 107 CFU mL-1 ], albeit with a slower action, which supports the involvement of specific virulence factors (e.g. chitinases, Fit toxin) to overcome the intestinal barrier to the haemocoel. Varying levels of bacterial virulence were observed on diverse target Diptera and Lepidoptera. CONCLUSION: The soil-dwelling bacterium P. protegens appears to have evolved its own potential as a stand-alone entomopathogen, yet the establishment of an opportunistic association with entomoparasitic nematodes would represent a special competitive advantage. This finding contributes to a deeper understanding of the nematode-bacteria biocontrol agent complex and the deriving paradigm of their use as biological control agents. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

The Mechanism of Chlorantraniliprole Resistance and Detoxification in Trichogramma chilonis (Hymenoptera: Trichogrammatidae).

Parasitic Trichogramma chilonis Ishii, an egg parasitoid of Grapholita molesta, is a critical agent for biological control of insect pests in crop plants. However, the efficiency of T. chilonis is influenced by its resistance to the common pesticide chlorantraniliprole. To elucidate the chlorantraniliprole detoxification mechanism, differentially expressed genes (DEGs) related to chlorantraniliprole resistance were studied at different developmental stages of the wasp. Individuals of T. chilonis were grouped and treated with chlorantraniliprole at different developmental stages. Untreated wasps were used as controls. Transcriptomic analysis identified the DEGs associated with chlorantraniliprole resistance and detoxification in T. chilonis. A total of 1,483 DEGs were associated with chlorantraniliprole resistance at all developmental stages. DEGs that correlated with chlorantraniliprole sensitivity of T. chilonis at different developmental stages were distinct and had various functions. The newly identified DEGs are involved in cytochrome P450- and glutathione metabolism-related pathways, which were predicted to contribute to chlorantraniliprole detoxification. Chlorantraniliprole detoxification by T. chilonis was associated with cytochrome P450- and glutathione-related pathways. Our findings may be useful for balancing chemical and biological control practices aimed to optimize agricultural production.

Parasitoids of Lobesia botrana (Lepidoptera: Tortricidae) in the Douro Demarcated Region vineyards and the prospects for enhancing conservation biological control.

The more restrictive regulations of pesticides in Europe have led to an increase in conservation biological control (CBC) research. However, little attention has been paid to the main determinants of Lobesia botrana parasitism. The Douro Demarcated Region landscape offers scope for the use of CBC. The study was conducted between 2002 and 2015 aiming at: (i) identifying parasitoids associated with L. botrana and evaluating their impact as biological control agents in each generation of the pest, and (ii) evaluating the effect of both the proportion of ecological infrastructures (EI) near the vineyards, and the impact of management practices (chemical treatments and ground cover) on the parasitism of L. botrana. A total of 3226 larvae/pupae of L. botrana were collected (15% were parasitized and 485 parasitoids emerged). A complex of 16 taxa of parasitoids was identified, the majority belonging to Hymenoptera. The most abundant were Elachertus sp. (Eulophidae), Campoplex capitator Aubert (Ichneumonidae), and Brachymeria tibialis (Walker) (Chalcididae), which represented 62.5, 12.6, and 12.0% of the total assemblage of parasitoids which emerged, respectively. The percentage of parasitism ranged from 0.0 to 61.5% (first generation), from 0.0 to 36.8% (second generation), and from 0.0 to 12.1% (third generation). Importantly, it was found that the parasitism rate was higher in vineyards with ground cover. In addition, EI in the area surrounding the vineyards produced a significant increase in parasitism. These results suggest potential for CBC of L. botrana if EI around vineyards, and ground cover with native perennial plants within vineyards, are encouraged.

Biology, parasitoid complex and potential distribution of saxaul's dominant defoliators, Teia dubia (Lepidoptera: Lymantriidae).

Natural enemies that impact pest populations must be understood in order to build integrated pest control strategies and to understand the most important aspects affecting pest dynamics. Haloxylon ammodendron (C. A. Mey.) Bunge is an important perennial plant species extensively used in sand stabilization and wind prevention in arid areas. This study aimed to determine the main defoliators that damage H. ammodendron and the parasitoid complex associated with them. Twelve species of defoliators were found in Northern Xinjiang, and Teia dubia (Tauscher) (Lepidoptera: Lymantriidae), Scrobipalpa sp. (Lepidoptera: Gelechiidae), and Eucharia festiva Hüfnagel (Lepidoptera: Arctiidae) were the dominant pests. T. dubia is the predominant defoliator with three generations a year. Northwest China, Central Asia, and the Mediterranean region are potentially suitable habitats for T. dubia in the world, while Xinjiang is the primary distribution area in China. Parasitoids belonging to seven species and four families were reared from the larvae of T. dubia, they were all endoparasitoids and koinobiont. Cotesia sp. (Hymenoptera: Braconidae) is the dominant parasitoid and prefer to parasitic in the 3rd-5th instar larvae. The present study provides the basis for understanding the species composition and natural enemies of lepidopteran defoliators. It will be an effective tool for the integrated pest management programs of H. ammodendron forest.

A teratocyte-specific serpin from the endoparasitoid wasp Cotesia vestalis inhibits the prophenoloxidase-activating system of its host Plutella xylostella.

Many endoparasitoids adopt several parasitic factors, such as venom, polydnavirus and teratocytes, to suppress the immune response of their associated hosts including melanization for successful parasitism. A teratocyte-specific expressed serpin gene, designated as CvT-serpin6, was identified from the parasitoid Cotesia vestalis. The immunoblot result suggested that CvT-serpin6 was secreted into extracellular space. qPCR results showed that CvT-serpin6 was mainly transcribed at later stages of parasitism, and the transcriptional abundance of CvT-serpin6 in teratocytes was significantly increased in response to the challenge of bacteria. Inhibitory assay indicated that recombinant CvT-serpin6 (rCvT-serpin6) could inhibit the activation of Plutella xylostella prophenoloxidase and ultimately resulted in the inhibition of melanization in P. xylostella haemolymph. Furthermore, we confirmed that rCvT-serpin6 could form SDS-stable complexes with activated PxPAP1 and PxPAP3 in a dose-dependent manner. Altogether, our results further shed insight into the molecular mechanisms that teratocytes involved in controlling host immune response.

Temperature-dependent modelling and spatial prediction reveal suitable geographical areas for deployment of two Metarhizium anisopliae isolates for Tuta absoluta management.

Tuta absoluta is one of the most devastating pests of Solanaceae crops in Africa. We previously demonstrated the efficacy of Metarhizium anisopliae isolates ICIPE 18, ICIPE 20 and ICIPE 665 against adult T. absoluta. However, adequate strain selection and accurate spatial prediction are fundamental to optimize their efficacy and formulations before field deployment. This study therefore assessed the thermotolerance, conidial yield and virulence (between 15 and 35 °C) of these potent isolates. Over 90% of conidia germinated at 20, 25 and 30 °C while no germination occurred at 15 °C. Growth of the three isolates occurred at all temperatures, but was slower at 15, 33 and 35 °C as compared to 20, 25 and 30 °C. Optimum temperatures for mycelial growth and spore production were 30 and 25 °C, respectively. Furthermore, ICIPE 18 produced higher amount of spores than ICIPE 20 and ICIPE 665. The highest mortality occurred at 30 °C for all the three isolates, while the LT50 values of ICIPE 18 and ICIPE 20 were significantly lower at 25 and 30 °C compared to those of ICIPE 665. Subsequently, several nonlinear equations were fitted to the mortality data to model the virulence of ICIPE 18 and ICIPE 20 against adult T. absoluta using the Entomopathogenic Fungi Application (EPFA) software. Spatial prediction revealed suitable locations for ICIPE 18 and ICIPE 20 deployment against T. absoluta in Kenya, Tanzania and Uganda. Our findings suggest that ICIPE 18 and ICIPE 20 could be considered as effective candidate biopesticides for an improved T. absoluta management based on temperature and location-specific approach.

Bracoviruses recruit host integrases for their integration into caterpillar's genome.

Some DNA viruses infect host animals usually by integrating their DNAs into the host genome. However, the mechanisms for integration remain largely unknown. Here, we find that Cotesia vestalis bracovirus (CvBV), a polydnavirus of the parasitic wasp C. vestalis (Haliday), integrates its DNA circles into host Plutella xylostella (L.) genome by two distinct strategies, conservatively and randomly, through high-throughput sequencing analysis. We confirmed that the conservatively integrating circles contain an essential "8+5" nucleotides motif which is required for integration. Then we find CvBV circles are integrated into the caterpillar's genome in three temporal patterns, the early, mid and late stage-integration. We further identify that three CvBV-encoded integrases are responsible for some, but not all of the virus circle integrations, indeed they mainly participate in the processes of early stage-integration. Strikingly, we find two P. xylostella retroviral integrases (PxIN1 and PxIN2) are highly induced upon wasp parasitism, and PxIN1 is crucial for integration of some other early-integrated CvBV circles, such as CvBV_04, CvBV_12 and CvBV_24, while PxIN2 is important for integration of a late-integrated CvBV circle, CvBV_21. Our data uncover a novel mechanism in which CvBV integrates into the infected host genome, not only by utilizing its own integrases, but also by recruiting host enzymes. These findings will strongly deepen our understanding of how bracoviruses regulate and integrate into their hosts.

Potential of in vivo- and in vitro-cultured entomopathogenic nematodes to infect Lobesia vanillana (Lepidoptera: Tortricidae) under laboratory conditions.

Entomopathogenic nematodes (EPNs) have been successfully applied as biological control agents against above ground and soil stages of insect pests. However, for commercial application, it is crucial to mass culture these nematodes using in vitro liquid culture technology, as it is not attainable when using susceptible insects as hosts. Lobesia vanillana (Lepidoptera: Tortricidae) is regarded a sporadic pest of wine grapes in South Africa. The in vivo- and in vitro-cultured South African EPNs, Steinernema yirgalemense and Steinernema jeffreyense (Rhabditida: Steinernematidae), were evaluated against larvae and pupae of L. vanillana in laboratory bioassays. For larvae, high mortality was observed for all treatments: In vitro-cultured S. yirgalemense (98%) performed better than S. jeffreyense (73%), while within in vivo cultures, there was no difference between nematode species (both 83%). No significant difference was detected between in vivo- and in vitro cultures of the same nematode species. The LD50 of the in vitro-cultured S. yirgalemense, was 7.33 nematodes per larva. Mortality by infection was established by dissecting L. vanillana cadavers and confirming the presence of nematodes, which was > 90% for all treatments. Within in vitro cultures, both S. yirgalemense and S. jeffreyense were able to produce a new cohort of infective juveniles from L. vanillana larvae. Pupae, however, were found to be considerably less susceptible to EPN infection. This is the first study on the use of EPNs to control L. vanillana. The relative success of in vitro-cultured EPN species in laboratory assays, without any loss in pathogenicity, is encouraging for further research and development of this technology.

Performance of Trichogramma japonicum under field conditions as a function of the factitious host species used for mass rearing.

Different factitious hosts were used to mass rear Trichogramma japonicum Ashmead in different parts of the globe because thorough details were lacking in both the laboratory and the field. The objective of this study was to compare, parasitoid, T. japonicum reared in different factitious hosts. Three commonly used factitious host eggs, Corcyra cephalonica (Stainton), Ephestia kuehniella Zeller and Sitotroga cerealella Olivier were tested under laboratory conditions and then in the field over a yellow stem borer, Scirpophaga incertulus (Walker) of rice. The highest parasitism by T. japonicum was observed on E. kuehniella eggs. The parasitoid's highest emergence (88.99%) was observed on S. cerealella eggs at 24 h exposure, whereas at 48 h it was on E. kuehniella eggs (94.66%). Trichogramma japonicum females that emerged from E. kuehniella eggs were significantly long-lived. The days of oviposition by hosts and the host species were significant individually, but not their interaction. Higher proportions of flying T. japonicum were observed when reared on E. kuehniella and C. cephalonica eggs. Field results showed that T. japonicum mass-reared on E. kuehniella showed higher parasitism of its natural host, S. incertulus eggs. Hence, by considering these biological characteristics and field results, E. kuehniella could be leveraged for the mass rearing of quality parasitoids of T. japonicum in India, the Asian continent and beyond.

Cold storage-mediated rearing of Trichogramma evanescens Westwood on eggs of Plodia interpunctella (Hübner) and Galleria mellonella L.

The egg parasitoid Trichogramma evanescens Westwood is considered as an efficient biological control agent for managing several lepidopteran pests and it is widely distributed throughout the world. Mass rearing protocols of parasitoids that are currently in use in biocontrol programs require a meticulous quality control plan, in order to optimize their efficacy, but also their progeny production capacity. In this paper, the effect of different factors on the quality control in mass rearing of T. evenescens, using Plodia interpunctella (Hübner) and Galleria mellonella L. as host species, were investigated. The impact of egg agewas significant in the rates of parasitism, for both host species tested. Significantly highest percent of parasitoid emergence was noticed in two day-old eggs for both host species, while one day-old eggs day exhibited the maximum emergence when both species were used togetherin the same trials. Age-dependent storage egg preservation at either 4 or 9°C significantly influenced the parasitism percentages on both species. The highest parasitism percentage was recorded in two day-old G. mellonella eggs that are kept for 15 days at 9°C while the lower in one day-old P. interpunctella eggs for 60 d storage. Moreover, the highest parasitoid mortality was recorded in T. evanescens reared either on P. interpunctella or G. mellonella at 20°C. Rearing of the parasitoid on a mixture of eggs of both host species resulted in higher parasitism, but not always in higher rates of parasitoid emergence. The results of the present work provide useful information that can be further utilized in rearing protocols of T. evanescens.

Conservative production of galactosaminogalactan in Metarhizium is responsible for appressorium mucilage production and topical infection of insect hosts.

The exopolysaccharide galactosaminogalactan (GAG) has been well characterized in Aspergilli, especially the human pathogen Aspergillus fumigatus. It has been found that a five-gene cluster is responsible for GAG biosynthesis in Aspergilli to mediate fungal adherence, biofilm formation, immunosuppression or induction of host immune defences. Herein, we report the presence of the conserved GAG biosynthetic gene cluster in the insect pathogenic fungus Metarhizium robertsii to mediate either similar or unique biological functions. Deletion of the gene cluster disabled fungal ability to produce GAG on germ tubes, mycelia and appressoria. Relative to the wild type strain, null mutant was impaired in topical infection but not injection of insect hosts. We found that GAG production by Metarhizium is partially acetylated and could mediate fungal adherence to hydrophobic insect cuticles, biofilm formation, and penetration of insect cuticles. In particular, it was first confirmed that this exopolymer is responsible for the formation of appressorium mucilage, the essential extracellular matrix formed along with the infection structure differentiation to mediate cell attachment and expression of cuticle degrading enzymes. In contrast to its production during A. fumigatus invasive growth, GAG is not produced on the Metarhizium cells harvested from insect hemocoels; however, the polymer can glue germ tubes into aggregates to form mycelium pellets in liquid culture. The results of this study unravel the biosynthesis and unique function of GAG in a fungal system apart from the aspergilli species.