Characterization of the serosal cells surrounding Cotesia kariyai larvae and their role in host immunosuppression.

About half of the serosal cells (Scs) of Cotesia kariyai (Ck) eggs are released as teratocytes into the host body cavity after hatching, and another half remain attached to the larval epidermis until the 1st instar larvae of Ck ecdysis to 2nd instars. To investigate the role of the serosal cells surrounding Ck 1st instar larvae (1st Scs) in immune avoidance, Ck 1st instar larvae with and without Scs removed using dispase were transplanted into Mythimna separata larvae (MsLrv), respectively. As a result, Ck 1st instar larvae surrounded by Scs were less susceptible to the MsL encapsulation than Ck 1st instar larvae without the Scs, suggesting that the Scs are involved in suppressing the encapsulation of the MsL. Furthermore, when the granular cells and plasmatocytes of the MsL involved in the encapsulation were incubated in a medium supplemented with proteins extracted from 1st Scs, the plasmatocytes failed to adhere to glass slides, and did not spread their filopodium and lamellipodium. These findings suggest that 1st Scs express proteins that inhibit filopodium and lamellipodium spreading to prevent the MsL plasmatocytes from adhering to Ck larvae.

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.

Geographic Distribution, Genetic Variability and Biological Properties of Rice Orange Leaf Phytoplasma in Southeast Asia.

Rice orange leaf phytoplasma (ROLP) causes clear orange to yellowish leaf discoloration and severe stunting in rice seedlings. The ecological and biological characteristics of ROLP are largely unknown because the disease has not widely caused serious problems in rice cultivated areas, thereby leading to the low accumulation of research data. However, in the past decade, the disease became a threat to rice production, particularly in South China and India; it has also been recognised in other Asian countries, such as Vietnam, Thailand and the Philippines. Here, we observed the occurrence of ROLP in paddies of the Southeast Asian counties (Cambodia, Vietnam and the Philippines) and found that the isolates in the Philippines and Vietnam were monophyletic, while those in India, Thailand and Cambodia were more diverse, suggesting their potential origins. In Cambodia, it was revealed that following polymerase chain reaction (PCR) detection, the known ROLP-insect vectors, N. virescens Distant and Recilia dorsalis Motchulsky, were ROLP-positive, indicating their roles in pathogen dispersal. Moreover, fluorescent and scanning electron microscopy revealed the intensive accumulation of the phytoplasma in phloem tissues and massive accumulation of storage starch in vascular bundle sheath and parenchyma. Altogether, this study illustrated the genetic variability of global ROLP isolates and the pathogen's biological impact on rice tissue.

Reemerging Rice Orange Leaf Phytoplasma with Varying Symptoms Expressions and Its Transmission by a New Leafhopper Vector-Nephotettix virescens Distant.

Rice orange leaf phytoplasma (ROLP) belongs to the "Candidatus Phytoplasma asteris" 16SrI-B subgroup, which is solely transmitted by the zigzag-striped leafhopper (Recilia dorsalis Motchulsky) and the green leafhopper (Nephotettix cincticeps Uhler) (Hemiptera: Cicadellidae). Recently, rice plants showing orange leaf discoloration have become ubiquitous in several paddies of two provinces in the Philippines. In total of 98 symptomatic rice plants, 82% (Laguna) and 95% (Mindanao) were ROLP-positive by nested PCR detection. These plants showed more varying symptoms than previously reported. The vector insect R. dorsalis was scarcely present but green paddy leafhopper, N. virescens Distant (Hemiptera: Cicadellidae), was commonly observed in the paddies, thus the ability of N. virescens to transmit ROLP was thoroughly investigated. Newly emerged adult N. virescens, which fed on ROLD-source rice plants, were used to inoculate a susceptible rice seedling and was serially transferred into a new healthy seedling. Resultant positive transmission rates varied from 5.1% to 17.8%. The transmission ability of the insects was generally decreased over time. These findings suggest that N. virescens is an alternative vector of ROLP in the Philippines. Altogether, this study highlighted the increasing importance of ROLD-reemergence in Southeast and East Asia and proved the need for careful management of this alternative vector insect.

Viral infection induces different detoxification enzyme activities in insecticide-resistant and -susceptible brown planthopper Nilaparvata lugens strains.

This study aimed to describe the relationship between viral infection in Nilaparvata lugens (Stål), the brown planthopper (BPH), and different insecticide susceptibilities. BPH-resistant strains were selected using fenthion (an organophosphate) or etofenprox (a pyrethroid); a susceptible strain was used as the baseline colony before insecticide selection. All strains were infected with rice ragged stunt virus (RRSV) or rice grassy stunt virus (RGSV), after which the activities of three detoxification enzymes, cytochrome-P450-monooxygenase (P450), glutathione S-transferase (GST), and carboxylesterase (CE), were compared. Males of the strains selected for both insecticides showed high P450 and GST-CDNB activities. The activity of all enzymes was higher in males than in females, as a whole. However, males of the susceptible strain infected with RRSV showed decreased CE and GST-CDNB activities. BPH with low susceptibility to etofenprox showed a marked increase in P450 activity after RRSV infection; the GST-CDNB activity of females in the insecticide-resistant strain increased. RGSV infection induced high CE and P450 activities in etofenprox-selected females. The RRSV infection rate, but not the RGSV, decreased in etofenprox-selected strains.

Regional population differences of the brown planthopper (Nilaparvata lugens Stål) in Cambodia using genotyping-by-sequencing.

The brown planthopper Nilaparvata lugens Stål (BPH) can be found year-round in tropical region and causes severe damage to rice. Although there has been documented BPH damage to rice crops in the past decade in Cambodia, the extent of this epidemic is poorly understood. Here, we examined the time variation of BPH population in the abundance of morphotypes in 13 main rice-producing provinces (86 sites) by aspirator method and in the Takeo Province (five sites) by yellow sticky trap method. At least three generations were observed during the 3-month collection period in the rainy growing season. Regarding the occurrence of BPH morphotypes, in July the macropterous adults were restricted to south Cambodia and in August all morphotypes, adults (macropterous and brachypterous) and nymphs, appeared in all sampling sites. To explain the difference of regional distribution, the genetic differentiation was analyzed in south and northwest Cambodia (three sites) by using single nucleotide polymorphisms (SNP) analysis via genotyping-by-sequencing (GBS) using next-generation sequencing. The 2455 SNPs obtained by GBS clarified the three sub-populations and they corresponded to the expected dissemination patterns. These results provide a clue to understand the differentiation and epidemic of BPH in Cambodia.

Potential Host Range of the Larval Endoparasitoid Cotesia vestalis (=plutellae) (Hymenoptera: Braconidae).

Many parasitoid wasps are highly specialized in nature, attacking only one or a few species of hosts. Host range is often determined by a range of biological and ecological characteristics of the host including diet, growth potential, immunity, and phylogeny. The solitary koinobiont endoparasitoid wasp, Cotesia vestalis, mainly parasitizes diamondback moth (DBM) larvae in the field, although it has been reported that to possess a relatively wide lepidopteran host range. To better understand the biology of C vestalis as a potential biological control of hosts other than the DBM, it is necessary to determine suitability for potential hosts. In this study, the potential host range of the wasp and its developmental capacity in each host larva were examined under laboratory conditions using 27 lepidopteran species from 10 families. The wasp was able to parasitize 15 of the 27 species successfully. Some host species were not able to exclude C vestalis via their internal physiological defenses. When parasitization was unsuccessful, most hosts killed the parasitoid at the egg stage or early first-instar stage using encapsulation, but some host species disturbed the development of the parasitoid at various stages. No phylogenetic relationships were found among suitable and unsuitable hosts, revealing that host range in some endoparasitoids is not constrained by relatedness among hosts based on immunity.

Current status of insecticide susceptibility in the brown planthopper in Cambodia.

Little information exists about pesticide availability and its effect on pest control in rural regions of developing countries. The availability of different insecticidal ingredients in rural areas of Cambodia was determined by inspecting labels on products used by farmers and in retail shops. A large number of products available in markets and used by farmers contained abamectin, emamectin benzoate, cypermethrin, and chlorpyrifos. The effects on the brown planthopper (BPH; Nilaparvata lugens (Stål)) were investigated by comparing the susceptibility of three BPH populations in Cambodia to twelve active ingredients in 2015. All populations showed high susceptibility to abamectin and cypermethrin; however, regional differences in susceptibility were observed for the other ingredients. The implication was that farmers selected the most effective products based on sellers' opinions. It is important to monitor insecticide use and BPH susceptibility in each region of Cambodia in order to minimize the risk of high BPH population densities.

WASP-ASSOCIATED FACTORS ACT IN INTERSPECIES COMPETITION DURING MULTIPARASITISM.

Coexistence or displacement of parasitoids in hosts during intrinsic competitive interactions between different parasitoid species (multiparasitism) may depend on their life history traits and behavior. Intense competition for possession of hosts may lead to the elimination of the inferior competitor through physical attack and/or physiological suppression. However, the mechanisms of physiological suppression during multiparasitism remain unclear. Previous work has shown that first instar larvae of the solitary endoparasitoid Meteorus pulchricornis possess well-developed mandibles that are used to kill competitors. Two gregarious endoparasitoids, Cotesia kariyai and C. rufricus, share host resources especially when the time gap of oviposition is short. Here, we investigated the physiological influence of wasp-regulatory factors of the three endoparasitoids, M. pulchricornis, C. kariyai, and C. ruficrus, in their common host Mythimna separata. We found that MpVLP alone (or with venom) deleteriously affected the development of the two gregarious species. Similarly, CkPDV plus venom had toxic effect on M. pulchricornis eggs and immature larvae, although they were not harmful to immature stages of C. ruficrus. Cotesia kariyai and C. ruficrus were able to coexist mainly through the expression of regulatory factors and both could successfully emerge from a multiparasitized host. The injection of CkPDV plus venom after oviposition in L5 host larvae facilitated C. ruficrus development and increased the rate of successful parasitism from 9% to 62%. This suggests that the two gregarious parasitoid wasps exhibit strong phylogenetic affinity, favoring their coexistence and success in multiparasitized hosts.

Differential Juvenile Hormone Variations in Scale Insect Extreme Sexual Dimorphism.

Scale insects have evolved extreme sexual dimorphism, as demonstrated by sedentary juvenile-like females and ephemeral winged males. This dimorphism is established during the post-embryonic development; however, the underlying regulatory mechanisms have not yet been examined. We herein assessed the role of juvenile hormone (JH) on the diverging developmental pathways occurring in the male and female Japanese mealybug Planococcus kraunhiae (Kuwana). We provide, for the first time, detailed gene expression profiles related to JH signaling in scale insects. Prior to adult emergence, the transcript levels of JH acid O-methyltransferase, encoding a rate-limiting enzyme in JH biosynthesis, were higher in males than in females, suggesting that JH levels are higher in males. Furthermore, male quiescent pupal-like stages were associated with higher transcript levels of the JH receptor gene, Methoprene-tolerant and its co-activator taiman, as well as the JH early-response genes, Krüppel homolog 1 and broad. The exposure of male juveniles to an ectopic JH mimic prolonged the expression of Krüppel homolog 1 and broad, and delayed adult emergence by producing a supernumeral pupal stage. We propose that male wing development is first induced by up-regulated JH signaling compared to female expression pattern, but a decrease at the end of the prepupal stage is necessary for adult emergence, as evidenced by the JH mimic treatments. Furthermore, wing development seems linked to JH titers as JHM treatments on the pupal stage led to wing deformation. The female pedomorphic appearance was not reflected by the maintenance of high levels of JH. The results in this study suggest that differential variations in JH signaling may be responsible for sex-specific and radically different modes of metamorphosis.

Prophenoloxidase genes and antimicrobial host defense of the model beetle, Tribolium castaneum.

In this study, we characterized prophenoloxidase (proPO, (PPO)) genes of Tribolium castaneum and examined their involvement in antimicrobial host defense. Amino acid sequence comparison with well-characterized PPO proteins from other insect species suggested that T. castaneum PPO genes encoded functional proenzymes, with crucial sequence motifs being conserved. Developmental kinetics of the mRNA of two PPO genes, PPO1 and PPO2 in the pupal stage were different to each other. The PPO1 mRNA levels consistently decreased during pupal development while that of PPO2 peaked at mid-pupal stage. The two mRNAs also exhibited distinct responses upon immune challenges with heat-killed model microbes. The PPO1 mRNA stayed nearly unchanged by 6h post challenge, and was somewhat elevated at 24h. In contrast, the PPO2 mRNA significantly decreased at 3, 6 and 24h post challenge. These trends exhibited by respective PPO genes were consistent irrespective of the microbial species used as elicitors. Finally, we investigated the involvement of T. castaneum PPO genes in antimicrobial host defense by utilizing RNA interference-mediated gene silencing. Survival assays demonstrated that double knockdown of PPO genes, which was accompanied by weakened hemolymph PO activities, significantly impaired the host defense against Bacillus subtilis. By contrast, the knockdown did not influence the induction of any of the T. castaneum antimicrobial peptide genes that were studied here, except for one belonging to the gene group that shows very weak or negligible microbial induction. PPO knockdown as well weakened host defense against Beauveria bassiana moderately but significantly depending on the combination of infection methods and targeted genes. Our results indicated that the PPO genes represented constituents of both antibacterial and antifungal host defense of T. castaneum.

Peptidoglycan recognition protein genes and their roles in the innate immune pathways of the red flour beetle, Tribolium castaneum.

We have previously demonstrated that the functional Toll and IMD innate immune pathways indeed exist in the model beetle, Tribolium castaneum while the beetle's pathways have broader specificity in terms of microbial activation than that of Drosophila. To elucidate the molecular basis of this broad microbial activation, we here focused on potential upstream sensors of the T. castaneum innate immune pathways, peptidoglycan recognition proteins (PGRPs). Our phenotype analyses utilizing RNA interference-based comprehensive gene knockdown followed by bacterial challenge suggested: PGRP-LA functions as a pivotal sensor of the IMD pathway for both Gram-negative and Gram-positive bacteria; PGRP-LC acts as an IMD pathway-associated sensor mainly for Gram-negative bacteria; PGRP-LE also has some roles in Gram-negative bacterial recognition of the IMD pathway. On the other hand, we did not obtain clear phenotype changes by gene knockdown of short-type PGRP genes, probably because of highly inducible nature of these genes. Our results may collectively account for the promiscuous bacterial activation of the T. castaneum innate immune pathways at least in part.

Expressional and functional analysis of CYP15A1, a juvenile hormone epoxidase, in the red flour beetle Tribolium castaneum.

Juvenile hormone (JH) is synthesized and secreted by the corpora allata. In the final two steps of JH biosynthesis, farnesoic acid (FA) is converted to JH through methylation by JH acid O-methyltransferase (JHAMT) and epoxidation by the cytochrome P450 enzyme CYP15. In the present study, we identified a homolog of CYP15 from the red flour beetle Tribolium castaneum (TcCYP15A1), and analyzed its expression as well as its role in JH biosynthesis. Quantitative RT-PCR analysis showed that the level of TcCYP15A1 mRNA was high in the embryonic stage as well as in the middle of the final larval instar. In the embryonic stage, the transcript level of TcCYP15A1 started to increase 30h after egg laying (AEL), peaked 54-60h AEL, and was followed by an increase of TcJHAMT mRNA, suggesting that JH biosynthesis started at this time point. TcCYP15A1 mRNA was present, but not exclusively so in the larval corpora allata. The recombinant TcCYP15A1 protein epoxidized both FA and methyl farnesoate (MF) in highly stereo-specific manners. These results confirmed that TcCYP15A1 is involved in JH biosynthesis. The RNAi-mediated knockdown of TcCYP15A1 in the pre-final larval instar did not result in precocious metamorphosis to pupa, indicating that MF may exhibit JH-like activity in order to maintain the larval status. The double knockdown of TcJHAMT and TcCYP15A1 resulted in pupae and adults with shorter wings, suggesting that the precursors of JH, JH acid and MF, may be essential for wing expansion.

A novel type of hemocytes localizing melanization with high-spreading behavior in Mythimna separata.

Lepidopteran larvae show a cellular response to invading foreign substances that are larger than hemocytes, for example, parasitoid eggs or larvae. This response is called hemocyte encapsulation and is often accompanied by phenoloxidase (PO)-catalyzed melanization. In the present study, we artificially transplanted endoparasitoid larvae and small glass fragments into the hemocoel of the common armyworm, Mythimna separata. We observed that the host larva showed a cellular response and that, 2-4 h after transplantation, melanin formation was spatially confined to the surface of the encapsulated substances. We further noted that specific morphological hemocytes surrounded by melanin formation became attached to the surface of the foreign substances. We designated these hemocytes hyperspread cells (HSCs) on the basis of their specific characteristics and circumferential spread. We confirmed the occurrence of prophenoloxidase (PPO)/phenoloxidase (PO) on the periphery of the HSCs and in the substance secreted around the HSCs by using anti-PPO antibody. We were unable to detect PPO-mRNA in HSCs by using in situ hybridization, although we showed that oenocytoids contained PPO-mRNA and PPO protein. We used light microscopy and scanning electron microscopy to discriminate five main types of circulating M. separata hemocytes. We observed that HSCs differed from plasmatocytes, but spread out well. Further, during the encapsulation process, HSCs appeared to provide a localized melanization spot on the surface of foreign invaders.

Inhibitory effects of the ethyl acetate extract from bulbs of Scilla scilloides on lipoxygenase and hyaluronidase activities.

CONTEXT: Scilla scilloides Druce (Liliaceae) is a folk medicine to treat dermal inflammation; however, the medicinal properties of this plant have not been completely established. OBJECTIVE: The current study investigates the potent anti-inflammatory effects of S. scilloides bulbs for its traditional usage using lipoxygenase and hyaluronidase as the inflammation model. To gain insight into the active constituents, nine homoisoflavones (1-9) were subsequently tested. MATERIALS AND METHODS: Lipoxygenase and hyaluronidase inhibition of ethyl acetate extract from the bulbs of this plant within 2000 µg/mL or homoisoflavones within 1000 µM were determined by colorimetric methods. RAW264.7 cells were incubated with 10 or 50 µM homoisoflavones plus lipopolysaccharide (LPS) for 24 h. The culture media were collected and analyzed for determination of the nitric oxide (NO) level by the colorimetric Griess method to measure the extent of inflammation. RESULTS: The extract exhibited inhibitory effects on lipoxygenase and hyaluronidase activities with IC50 values 31.5 and 169 µg/mL, respectively. Among the nine homoisoflavones tested, four (1 and 3-5) resulted in 79.3-97.9% higher lipoxygenase inhibition than 6.7-32.7% of the others at 500 µM. Calculated IC50 values indicated 5 as the compound responsible for strong lipoxygenase inhibition with 15.8 µM as the IC50 value. In the hyaluronidase assay, all homoisoflavones tested at 1000 µM demonstrated 16.2-58.0% inhibition. Incubating the cells in the presence of all nine homoisoflavones tested at 50 µM significantly suppressed the NO production, downward to 1.5-66.0%, in the LPS-activated macrophage cells as a model. DISCUSSION AND CONCLUSION: These results may indicate a potential role of S. scilloides for anti-inflammatory purposes.

Duplication of acetylcholinesterase gene in diamondback moth strains with different sensitivities to acephate.

This study examined the acetylcholinesterase 1 gene (AChE1) in Plutella xylostella strains with different sensitivities to acephate. Multiple haplotypes of the gene were found in the field-collected strains including distinct haplotypes carrying one or both previously reported mutations (A298S and G324A). Moreover, sequencing results indicated the presence of duplicated copies of the gene in the field-collected strains. No correlation was found between copy numbers of AChE1 and levels of resistance to acephate suggesting that extensive AChE1 duplication is not a major resistance factor at least in some P. xylostella strains. Proportions of the A298S and G324A mutations showed no correlation with levels of resistance to acephate. This suggests that acephate resistance of P. xylostella is complex and cannot be evaluated based on the AChE1 copy number or proportions of the resistance mutations alone.

Homoisoflavones as the antioxidants responsible from bulbs of Scilla scilloides.

Scilla scilloides Druce has been used as a folk medicine to treat dermal inflammation; however, the medicinal property of this plant remains to be entirely clarified. The ethyl acetate extract prepared from bulbs of S. scilloides exhibited antioxidative activities on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, hydrogen peroxide (H2O2) and nitric oxide (NO) scavenging assays. Nine homoisoflavones (1-9) yielded from this extract were further examined for their antioxidative activities. Among these chemicals tested, five homoisoflavones (1-3, 5 and 7), six homoisoflavones (1-3 and 5-7) and two homoisoflavones (4 and 5) resulted in showing higher activities than the others in DPPH radical, H2O2 and NO scavenging assays, respectively. Calculated EC50 values indicate 3 as the strongest in the DPPH radical scavenging analysis. These results may indicate a potential role of S. scilloides for its medicinal use and homoisoflavones as the antioxidants responsible.

Anti-inflammatory and antioxidative effects of a methanol extract from bulbs of Scilla scilloides.

The extract from bulbs of Scilla scilloides exhibited inhibitory effects in lipoxygenase and hyaluronidase assays and various oxidation models in vitro. Incubating the cells in the presence of this extract ameliorated t-butyl hydroperoxide-induced cytotoxicity from 27% to 57% in a macrophage model. The results may indicate the potential role of S. scilloides for its anti-inflammatory and antioxidative effects.

Five new nortriterpenoid glycosides from the bulbs of Scilla scilloides.

Five new norlanostane-type triterpenoid glycosides were isolated from the bulbs of Scilla scilloides DRUCE (Liliaceae). Their chemical structures were determined on the basis of spectroscopic data as well as chemical evidence.

Intrinsic inter- and intraspecific competition in parasitoid wasps.

Immature development of parasitoid wasps is restricted to resources found in a single host that is often similar in size to the adult parasitoid. When two or more parasitoids of the same or different species attack the same host, there is competition for monopolization of host resources. The success of intrinsic competition differs between parasitoids attacking growing hosts and parasitoids attacking paralyzed hosts. Furthermore, the evolution of gregarious development in parasitoids reflects differences in various developmental and behavioral traits, as these influence antagonistic encounters among immature parasitoids. Fitness-related costs (or benefits) of competition for the winning parasitoid reveal that time lags between successive attacks influence the outcome of competition. Physiological mechanisms used to exclude competitors include physical and biochemical factors that originate with the ovipositing female wasp or her progeny. In a broader multitrophic framework, indirect factors, such as plant quality, may affect parasitoids through effects on immunity and nutrition.