Glucose and trehalose metabolism through the cyclic pentose phosphate pathway shapes pathogen resistance and host protection in Drosophila.

Activation of immune cells requires the remodeling of cell metabolism in order to support immune function. We study these metabolic changes through the infection of Drosophila larvae by parasitoid wasp. The parasitoid egg is neutralized by differentiating lamellocytes, which encapsulate the egg. A melanization cascade is initiated, producing toxic molecules to destroy the egg while the capsule also protects the host from the toxic reaction. We combined transcriptomics and metabolomics, including 13C-labeled glucose and trehalose tracing, as well as genetic manipulation of sugar metabolism to study changes in metabolism, specifically in Drosophila hemocytes. We found that hemocytes increase the expression of several carbohydrate transporters and accordingly uptake more sugar during infection. These carbohydrates are metabolized by increased glycolysis, associated with lactate production, and cyclic pentose phosphate pathway (PPP), in which glucose-6-phosphate is re-oxidized to maximize NADPH yield. Oxidative PPP is required for lamellocyte differentiation and resistance, as is systemic trehalose metabolism. In addition, fully differentiated lamellocytes use a cytoplasmic form of trehalase to cleave trehalose to glucose and fuel cyclic PPP. Intracellular trehalose metabolism is not required for lamellocyte differentiation, but its down-regulation elevates levels of reactive oxygen species, associated with increased resistance and reduced fitness. Our results suggest that sugar metabolism, and specifically cyclic PPP, within immune cells is important not only to fight infection but also to protect the host from its own immune response and for ensuring fitness of the survivor.

Recognition of nonself is necessary to activate Drosophila's immune response against an insect parasite.

BACKGROUND: Innate immune responses can be activated by pathogen-associated molecular patterns (PAMPs), danger signals released by damaged tissues, or the absence of self-molecules that inhibit immunity. As PAMPs are typically conserved across broad groups of pathogens but absent from the host, it is unclear whether they allow hosts to recognize parasites that are phylogenetically similar to themselves, such as parasitoid wasps infecting insects. RESULTS: Parasitoids must penetrate the cuticle of Drosophila larvae to inject their eggs. In line with previous results, we found that the danger signal of wounding triggers the differentiation of specialized immune cells called lamellocytes. However, using oil droplets to mimic infection by a parasitoid wasp egg, we found that this does not activate the melanization response. This aspect of the immune response also requires exposure to parasite molecules. The unidentified factor enhances the transcriptional response in hemocytes and induces a specific response in the fat body. CONCLUSIONS: We conclude that a combination of danger signals and the recognition of nonself molecules is required to activate Drosophila's immune response against parasitic insects.

A parasitoid regulates 20E synthesis and antibacterial activity of the host for development by inducing host nitric oxide production.

Parasitoids are important components of the natural enemy guild in the biological control of insect pests. They depend on host resources to complete the development of a specific stage or whole life cycle and thus have evolved towards optimal host exploitation strategies. In the present study, we report a specific survival strategy of a fly parasitoid Exorista sorbillans (Diptera: Tachinidae), which is a potential biological control agent for agricultural pests and a pest in sericulture. We found that the expression levels of nitric oxide synthase (NOS) and nitric oxide (NO) production in host Bombyx mori (Lepidoptera: Bombycidae) were increased after E. sorbillans infection. Reducing NOS expression and NO production with an NOS inhibitor (NG-nitro-L-arginine methyl ester hydrochloride) in infected B. mori significantly impeded the growth of E. sorbillans larvae. Moreover, the biosynthesis of 20-hydroxyecdysone (20E) in infected hosts was elevated with increasing NO production, and inhibiting NOS expression lowered 20E biosynthesis. More importantly, induced NO synthesis was required to eliminate intracellular bacterial pathogens that presumably competed for shared host resources. Inhibiting NOS expression down-regulated the transcription of antimicrobial peptide genes and increased the number of bacteria in parasitized hosts. Collectively, this study revealed a new perspective on the role of NO in host-parasitoid interactions and a novel mechanism for parasitoid regulation of host physiology to support its development.

Shaping the Microbial Landscape: Parasitoid-Driven Modifications of Bactrocera dorsalis Microbiota.

Koinobiont endoparasitoids regulate the physiology of their hosts through altering host immuno-metabolic responses, processes which function in tandem to shape the composition of the microbiota of these hosts. Here, we employed 16S rRNA and ITS amplicon sequencing to investigate whether parasitization by the parasitoid wasps, Diachasmimorpha longicaudata (Ashmaed) (Hymenoptera: Braconidae) and Psyttalia cosyrae (Wilkinson) (Hymenoptera: Braconidae), induces gut dysbiosis and differentially alter the gut microbial (bacteria and fungi) communities of an important horticultural pest, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae). We further investigated the composition of bacterial communities of adult D. longicaudata and P. cosyrae to ascertain whether the adult parasitoids and parasitized host larvae share microbial taxa through transmission. We demonstrated that parasitism by D. longicaudata induced significant gut perturbations, resulting in the colonization and increased relative abundance of pathogenic gut bacteria. Some pathogenic bacteria like Stenotrophomonas and Morganella were detected in both the guts of D. longicaudata-parasitized B. dorsalis larvae and adult D. longicaudata wasps, suggesting a horizontal transfer of microbes from the parasitoid to the host. The bacterial community of P. cosyrae adult wasps was dominated by Arsenophonus nasoniae, whereas that of D. longicaudata adults was dominated by Paucibater spp. and Pseudomonas spp. Parasitization by either parasitoid wasp was associated with an overall reduction in fungal diversity and evenness. These findings indicate that unlike P. cosyrae which is avirulent to B. dorsalis, parasitization by D. longicaudata induces shifts in the gut bacteriome of B. dorsalis larvae to a pathobiont-dominated community. This mechanism possibly enhances its virulence against the pest, further supporting its candidacy as an effective biocontrol agent of this frugivorous tephritid fruit fly pest.

Regulating the expression of gene drives is key to increasing their invasive potential and the mitigation of resistance.

Homing-based gene drives use a germline source of nuclease to copy themselves at specific target sites in a genome and bias their inheritance. Such gene drives can be designed to spread and deliberately suppress populations of malaria mosquitoes by impairing female fertility. However, strong unintended fitness costs of the drive and a propensity to generate resistant mutations can limit a gene drive's potential to spread. Alternative germline regulatory sequences in the drive element confer improved fecundity of carrier individuals and reduced propensity for target site resistance. This is explained by reduced rates of end-joining repair of DNA breaks from parentally deposited nuclease in the embryo, which can produce heritable mutations that reduce gene drive penetrance. We tracked the generation and selection of resistant mutations over the course of a gene drive invasion of a population. Improved gene drives show faster invasion dynamics, increased suppressive effect and later onset of target site resistance. Our results show that regulation of nuclease expression is as important as the choice of target site when developing a robust homing-based gene drive for population suppression.

Honey bee (Apis mellifera) nurse bee visitation of worker and drone larvae increases Varroa destructor mite cell invasion.

The life cycle of Varroa destructor, the ectoparasitic mite of honey bees (Apis mellifera), includes a dispersal phase, in which mites attach to adult bees for transport and feeding, and a reproductive phase, in which mites invade worker and drone brood cells just prior to pupation to reproduce while their bee hosts complete development. In this study, we wanted to determine whether increased nurse bee visitations of adjacent drone and worker brood cells would increase the likelihood of Varroa mites invading those cells. We also explored whether temporarily restricting the nurses' access to sections of worker brood for 2 or 4 h would subsequently cause higher nurse visitations, and thus, higher Varroa cell invasions. Temporarily precluding larvae from being fed by nurses subsequently led to higher Varroa infestation of those sections in some colonies, but this pattern was not consistent across colonies. Therefore, removing highly infested sections of capped worker brood could be further explored as a potential mechanical/cultural method for mite control. Our results provide more information on how nurse visitations affect the patterns of larval cell invasion by Varroa. Given that the mite's successful reproduction depends on the nurses' ability to visit and feed developing brood, more studies are needed to understand the patterns of Varroa mite invasion of drone and worker cells to better combat this pervasive honey bee parasite.

Comparative investigation of different chemicals on the germination of two aquatic microsporidia.

Microsporidia are eukaryotic obligatory intracellular parasites that infect a wide range of vertebrates and invertebrate hosts. Spores infect target cells of the host by transferring their sporoplasm through a distinctive polar tube. This study investigated how selected chemicals influence the germination of two newly discovered microsporidia species from central-western Iran. Spores of Parathelohania iranica were extracted from infected larvae of Anopheles superpictus s.l. and purified by the Percoll discontinuous density gradient method. Because of the small number of spores per copepod, extraction and purification were not performed for spores of the microsporidium infecting Paracyclops chiltoni. Both spores were exposed to KCl, NaCl, KI, NaI, and H2O2 and the effects of concentration (0.5, 1.5, and 2.5 M), pH (7.0, 9.0, and 11.0), temperature (4 °C and 25 °C), and duration of exposure (10 and 30 min) on spore germination were investigated and compared. This study indicated that the type of the ionic nature of the surrounding environment of spores plays an important role in the release of polar tubes of both microsporidia. Additionally, anions played a more significant role than cations. This effect was directly related to concentration, temperature, and time. However, no specific pattern was recognized at different alkaline pH levels. Hydrogen peroxide was not effective in releasing the polar tubes of the spores of these microsporidia. This study demonstrated the comparative role of some chemicals and the associated factors in the release of the polar tube of two aquatic microsporidia. Future research should examine the practical value of these findings in the mass production of candidate microsporidia for the biological control of pest invertebrate hosts.

Effects of minimum Strigea robusta (Digenea: Strigeidae) cercariae doses and localization of cysts on the anomaly P manifestation in Pelophylax lessonae (Anura: Ranidae) tadpoles.

The anomaly P is a mass morphological anomaly found in some populations of anuran amphibians (water frogs of the genus Pelophylax and toads of the genera Bufo and Bufotes) caused by the parasitic flatworm Strigea robusta. Minimum dose of cercariae for the appearance of the anomaly P remains unknown. However, it is important information for understanding of host population dynamics after invasion and the effects of the parasite on the second intermediate hosts. Herein, the invasion properties of S. robusta in Pelophylax lessonae tadpoles (Anura: Ranidae) and minimum dose for appearance of mild and severe forms of the anomaly P syndrome were described after direct experiments with certain numbers of cercariae exposure. Experimental groups of tadpoles have been exposed to eight doses of cercariae (2, 4, 6, 8, 10, 12, 14 and 16). A total of 63.8% tadpoles survived to the end of this experiment. It was revealed that a mild form of the anomaly P (polydactyly) can appear after infection by two cercariae, while the severe form traits appear after infection by four cercariae. The mean number of detected encysted metacercariae was reached to 53.5%. Differences in infection rates can be explained by the presence of an individual immune response in tadpoles or by the presence of different genetic lineages of the parasite infecting the same snail, which have different infectious potential. Low doses of infection leading to the induction of anomalies characterize S. robusta as a highly pathogenic species for amphibian species that are susceptible to infection and show an abnormal phenotype.

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.

Selective aspects of the epizootiology of Parathelohania iranica (Microsporidia: Amblyosporidae).

Parathelohania is a genus of microsporidia that preferentially attacks Anopheline mosquitoes. This study explored some selective aspects of the epizootiology of Parathelohania iranica (Microsporidia: Amblyosporidae) in its malaria mosquito host Anopheles superpictus s.l. (Diptera: Culicidae). For this study, Sar-rok Village, a place adjacent to the type locality of the parasite, located at the central western part of Iran was visited twice a month to collect host larvae from mid-summer to mid-autumn of 2017-2021. Patent infections were detected by the whitish discoloration of the involved segments. Superficially uninfected larvae were reared up to 26 days in the insectary to elucidate hidden infections. Molecular investigation and laboratory trials were conducted to evaluate the possibility of secondary infections in subsequent days. Morphological characters were used to determine the sex of larvae and adults. Data were entered in SPSS 23.0 and analyzed with relevant statistical tests as needed. In total, 584 P. iranica infected larvae of An. superpictus s.l. were collected in the study years at day zero (D0). Extended observations in the insectary revealed that 849 larvae (84.2 %), 22 dead pupae (2.2 %), and 137 emerged adults (13.6 %) were also infected. In the first two years of the study, the mean infection rate for D0 and D0 + D1-D26 infections was 6.25 % and 15.6 %, respectively. Exposure experiments indicated that subsequent infections in larvae (D1-D26) were not affected by a possible source in the accompanying field water. Patent (D0) infections were seasonal and had about a month delay compared to the general population of larvae. Concealed infections of larvae (D1-D26) were significantly more frequent in late mosquito season (P < 0.01). It is proposed that the gradual decrease of ambient temperature and the shortening of day length postpones the growth and development of P. iranica in the affected larvae. Both sexes of larvae were involved and none of them survived beyond a couple of days. The frequency of infections in adult males (5.8 %) were significantly more than females (4 %) (P = 0.02). The infections of larvae were more common in the late mosquito season, and the infections of adults were more frequent in the early mosquito season. This suggests the relative importance of vertical and horizontal routes of transmission in early and late mosquito seasons, respectively. However, age-specific data revealed that only 26 % of hidden infections of larvae (D1-D26) were stemmed from I to II age group. This implies that the vertical route may be less efficient than the horizontal route in the transmission of P. iranica to the mosquito host. These inferences should be verified with further field and laboratory investigations.

Two new species of Bacillidium (Microsporidia) from coelomocytes of Branchiura sowerbyi (Oligochaeta: Naididae) in China.

Bacillidium spp. exclusively infect oligochaetes and these microsporidian pathogens are typically characterized by their rod-shaped spores. Seven Bacillidium spp. are presently reported from different organs of oligochaetes. Here, we describe two new Bacillidium species, Bacillidium sinensis n. sp. and Bacillidium branchilis n. sp., from coelomocytes of Branchiura sowerbyi. This is the first report of Bacillidium spp. in oligochaetes from China. Both species of Bacillidium elicit the formations of opaque xenoma-like lesions in coelomocytes of the host. A diplokaryotic nucleus occurs in all life stages of these two new Bacillidium species. Mature spores of B. sinensis are 15.9 ± 0.6 (14.7-17.1) µm long (average ± standard error, range, n = 50) and 2.5 ± 0.1 (2.3-2.7) µm wide in fresh preparations. A new type of exospore (sixteen-layered exospore) is discovered from B. sinensis n. sp. which is distinctly different from B. branchilis n. sp., and other Bacillidium spp. (double-layered exospore) reported previously. These two Bacillidium species are morphologically distinguished from each other and all Bacillidium spp. described previously in terms of hosts, infection sites, spore size, spore wall or polar filament thickness. BLASTn searches indicated that these two new microsporidian parasites are surprisingly most similar to Janacekia tainanus (94.76% for B. sinensis and 90. 2% for B. branchilis) isolated from the fat body of midge larva (Kiefferulus tainanus). Phylogenetic analysis demonstrates that the two novel taxons cluster with J. debaisieuxi, J. tainanus, and Bacillidium sp. within the Jirovecia-Bacillidium-Janacekia clade. Other available 18S rRNA gene sequences for microsporidia that infect oligochaetes include J. sinensis, B. vesiculoformis, Neoflabelliforma aurantiae, and Bacillidium sp., but these do not form a single cluster with B. sinensis and B. branchilis, but are instead dispersed through the clade. Based on the ultrastructural features and molecular characteristics, two new species within the genus Bacillidium, B. sinensis n. sp. and B. branchilis n. sp., are designated.

Sublethal effects of organophosphorus insecticide phoxim on patch time allocation and oviposition behavior in a parasitoid wasp Meteorus pulchricornis.

Parasitoid wasps are key agents for controlling insect pests in integrated pest management programs. Although many studies have revealed that the behavior of parasitic wasps can be influenced by insecticides, the strategies of patch time allocation and oviposition have received less attention. In the present study, we forced the endoparasitoid Meteorus pulchricornis to phoxim exposure at the LC30 and tested the foraging behavior within patches with different densities of the host, the larvae of the tobacco cutworm Spodoptera litura. The results showed that phoxim treatment can significantly increase the patch-leaving tendency of female wasps, while host density had no impact. The number of oviposition and the number of previous patch visits also significantly influenced the patch time allocation decisions. The occurrence of oviposition behavior was negatively affected by phoxim exposure; however, progeny production was similar among patches with different host densities. Phoxim exposure shaped the offspring fitness correlates, including longer durations from cocoon to adult wasps, smaller body size, and shorter longevity. The findings of the present study highlight the sublethal effects that reduce the patch residence time and the fitness of parasitoid offspring, suggesting that the application of phoxim in association with M. pulchricornis should be carefully schemed in agroecosystems.

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.

Effect of Pepper Variety on the Susceptibility of Pepper Weevil Parasitoids.

Anthonomus eugenii Cano (Coleoptera: Curculionidae) is a key pest of cultivated peppers (Capsicum species) in tropical and subtropical America. Here we evaluated the effect of five pepper varieties on the susceptibility of A. eugenii to the parasitoids Bracon sp. (Hymenoptera: Braconidae), Eupelmus cushmani (Crawford) (Hymenoptera: Eupelmidae), and Jaliscoa hunteri Crawford (Hymenoptera: Pteromalidae). Potential parasitism was estimated by comparative analysis of parasitoid ovipositor size and the depth to which host larvae develop inside the fruit. Highest potential parasitism rates were achieved by Bracon sp. and E. cushmani on árbol and habanero peppers (84-99%) while the lowest rates were achieved by J. hunteri on serrano, bell, and jalapeño (7-18%). To validate potential parasitism rates, the actual parasitism rate by Bracon sp. and J. hunteri on three varieties of peppers was assessed. Actual parasitism rates of A. eugenii larvae in árbol were similar for Bracon sp. and J. hunteri (33%), while on bell and jalapeño Bracon sp. achieved 24% and 13% parasitism and J. hunteri achieved 14% and 8%, respectively. In most cases, actual parasitism was lower than estimated potential parasitism, although the latter had a notable predictive power (predicted R2 = 0.84). Results showed that the host was more vulnerable on small-fruited varieties because larvae were closer to the pericarp and could be reached by parasitoid ovipositors; likewise, in varieties with little placenta and seed, some larvae fed in the pericarp, where they were more vulnerable.

A Behavior-Manipulating Virus Relative as a Source of Adaptive Genes for Drosophila Parasitoids.

Some species of parasitic wasps have domesticated viral machineries to deliver immunosuppressive factors to their hosts. Up to now, all described cases fall into the Ichneumonoidea superfamily, which only represents around 10% of hymenoptera diversity, raising the question of whether such domestication occurred outside this clade. Furthermore, the biology of the ancestral donor viruses is completely unknown. Since the 1980s, we know that Drosophila parasitoids belonging to the Leptopilina genus, which diverged from the Ichneumonoidea superfamily 225 Ma, do produce immunosuppressive virus-like structure in their reproductive apparatus. However, the viral origin of these structures has been the subject of debate. In this article, we provide genomic and experimental evidence that those structures do derive from an ancestral virus endogenization event. Interestingly, its close relatives induce a behavior manipulation in present-day wasps. Thus, we conclude that virus domestication is more prevalent than previously thought and that behavior manipulation may have been instrumental in the birth of such associations.

The cost of travel: How dispersal ability limits local adaptation in host-parasite interactions.

Classical theory suggests that parasites will exhibit higher fitness in sympatric relative to allopatric host populations (local adaptation). However, evidence for local adaptation in natural host-parasite systems is often equivocal, emphasizing the need for infection experiments conducted over realistic geographic scales and comparisons among species with varied life history traits. Here, we used infection experiments to test how two trematode (flatworm) species (Paralechriorchis syntomentera and Ribeiroia ondatrae) with differing dispersal abilities varied in the strength of local adaptation to their amphibian hosts. Both parasites have complex life cycles involving sequential transmission among aquatic snails, larval amphibians and vertebrate definitive hosts that control dispersal across the landscape. By experimentally pairing 26 host-by-parasite population infection combinations from across the western USA with analyses of host and parasite spatial genetic structure, we found that increasing geographic distance-and corresponding increases in host population genetic distance-reduced infection success for P. syntomentera, which is dispersed by snake definitive hosts. For the avian-dispersed R. ondatrae, in contrast, the geographic distance between the parasite and host populations had no influence on infection success. Differences in local adaptation corresponded to parasite genetic structure; although populations of P. syntomentera exhibited ~10% mtDNA sequence divergence, those of R. ondatrae were nearly identical (<0.5%), even across a 900 km range. Taken together, these results offer empirical evidence that high levels of dispersal can limit opportunities for parasites to adapt to local host populations.

Multiple Attack to Inflorescences of an Annual Plant Does Not Interfere with the Attraction of Parasitoids and Pollinators.

Plants in the flowering stage need to ensure reproduction by protecting themselves from attack and by preserving interactions with mutualist pollinators. When different plant mutualists are using the same type of cues, such as volatile compounds, attraction of parasitoids and pollinators may trade off. To explore this, we compared volatile emission of Brassica nigra plants in response to single or dual attack on their inflorescences. Additionally, we recorded flower visitation by pollinators and the attraction of parasitoids in the greenhouse and/or field. Brassica nigra were exposed in the flowering stage to one or two of the following three attackers: Brevicoryne brassicae aphids, Pieris brassicae caterpillars, and Xanthomonas campestris pv. raphani bacteria. We found that single attack by caterpillars, and dual attack by caterpillars plus aphids, induced the strongest changes in plant volatile emission. The caterpillars' parasitoid C. glomerata did not exhibit preference for plants exposed to caterpillars only vs. plants exposed to caterpillars plus aphids or plus bacteria. However, the composition of the pollinator community associated with flowers of B. nigra was affected by plant exposure to the attackers, but the total number of pollinators visiting the plants did not change upon attack. We conclude that, when B. nigra were exposed to single or dual attack on their inflorescences, the plants maintained interactions with natural enemies of the insect attackers and with pollinators. We discuss how chemical diversity may contribute to plant resilience upon attack.

Effect of Leaf Maturity on Host Habitat Location by the Egg-Larval Parasitoid Ascogaster reticulata.

Adoxophyes honmai, a serious pest of tea plants, prefers to lay eggs on mature tea leaves rather than young leaves. Here, we examined a hypothesis that Ascogaster reticulata, an egg-larval parasitoid of A. honmai, increases the likelihood of encountering host egg masses by searching mature tea leaves when host-derived cues are not available. In a dual-choice bioassay using a four-arm olfactometer, A. reticulata preferred odor from intact, mature leaves versus young leaves. Based on volatile analysis with gas chromatography-mass spectrometry (GC-MS), we identified 5 and 10 compounds from mature and young leaf volatiles, respectively. The 5 components in the extract from intact mature leaves included (Z)-3-hexenyl acetate, (E)-ß-ocimene, linalool, (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT), and methyl salicylate. When each individual compound, or quaternary and quintenary blends of them, ratios of which were adjusted to match those of mature leaf volatiles, were provided, parasitoids preferred the full mixture and the quaternary blend devoid of DMNT to the solvent control. Methyl salicylate, one of the components of preferred blends, was not detected among young leaf volatiles. We concluded that the volatile composition of tea leaves changes, depending on their maturity, and that this composition affects foraging behavior of the parasitoid, which is closely related to the host herbivore's oviposition preference.

Abnormal increases in reactive oxygen species in dying insects infected with nematodes.

Stress enhances the concentration of reactive oxygen species (ROS) in animal plasma. Increased ROS alter various physiological functions, such as development and the immune response, but excessive increases could be harmful. In this study, we tested the hypothesis that abnormally increased plasma ROS levels are associated with animal death. Injection of the nematode Caenorhabditis elegans into insect larvae caused high mortality in Galleria mellonella, and the plasma ROS concentration was four times higher than M9 buffer-injected larvae. There was no difference in plasma antioxidant activity after nematode injection. However, coinjecting nematodes with an antioxidant (ascorbic acid or N-acetylcysteine) suppressed increases in ROS concentrations by the nematodes and increases in the number of nematodes in the larvae, which increased G. mellonella survival. These results suggest that the abnormal elevation of ROS associated with the stress caused by nematode propagation is lethal for G. mellonella.

Cky811 protein expressed by polydnavirus and venom gland of Cotesia kariyai regulates the host Mythimna separata larvae immune response function of C-type lectin responsible for foreign substance recognition which suppresses its melanization and encapsulation.

Cotesia kariyai (Ck) larvae implanted into the body cavity of the Mythimna separata (armyworm) larvae get melanized and encapsulated after adhesion by hemocytes called hyperspread cells (HSCs). The present study showed that HSCs could not adhere to the implanted Ck larvae in armyworm larvae after injection of Ck polydnavirus (CkPDV) + venom (V), thus melanization and encapsulation could not occur. A C-type lectin called Mys-IML of the host armyworm larvae was considered to be involved in the recognition of foreign substances which always expressed in hemocytes. The CkPDV DNA encodes a C-type lectin called Cky811 that has high amino acid homology to Mys-IML. HSCs did not adhere when CkPDV + V was mixed with the hemolymph of armyworm larvae on glass slides and incubated in vitro, but the addition of anti-Cky811 antibody enabled HSCs to adhere. The messenger RNA (mRNA) expression of Mys-IML in armyworm larvae injected with CkPDV + V became undetectable by 6 h. On the contrary, Cky811 mRNA was well expressed in the hemocytes of armyworm larvae injected with CkPDV + V from 0.5 to 6 h. Cky811 protein was also detected in the crude extracts from Ck venom gland + Ck venom reservoir, suggesting that these proteins regulate foreign substance recognition by the armyworm within 0.5 h. These results suggest that CkPDV + V suppresses mRNA expression of Mys-IML, and that Cky811 protein expressed in hemocytes regulates foreign substance recognition of Mys-IML, resulting in inhibition of the downstream reaction steps: HSCs adhesion, melanization, and encapsulation.