Widely targeted analysis of metabolomic changes of Cucumis sativus induced by cucurbit chlorotic yellows virus.

BACKGROUND: Plant metabolites play vital roles in regulating the behavior of herbivore insects. Virus infection can universally alter plant metabolites to manipulate the orientation and feeding behaviors of insect vector, to favor the transmission of virus. Thus, determining the differentially accumulated metabolites of plant upon virus infection could provide insights into understanding how the triple interactions among plant, virus and insect vector happens. Our previous studies have found that vector whitefly Bemisia tabaci (Gennadius, Hemiptera: Aleyrodidae) showed different orientation behavior and performance on CCYV-infected and healthy cucumber plants. Cucurbit chlorotic yellows virus (CCYV) is exclusively transmitted by B. tabaci in a semi-persistent mode. In this study, we take the CCYV, B. tabaci and cucumber as a research system to explore the functions of phyto-metabolites in the triple interactions. RESULTS: A total of 612 metabolites changed upon CCYV infection were monitored. Metabolites mainly enriched in flavonoids, lipids, nucleotides and their derivatives. At 7 days post CCYV inoculation (dpi), the contents of lipids, terpenoids and flavonoids remarkably decreased, while amino acids, nucleotides and their derivatives notably up-accumulated. At 15 dpi, the accumulation of flavonoids were still significantly reduced upon CCYV infection, while lipids, amino acids, nucleotides and derivatives were remarkably enhanced. Most of significantly increased metabolites were lipids (lysophosphatidylethanolamine, LPE; lysophosphatidylcholine, LPC and their isomers). Also, the number of significantly changed metabolites increased with the infection period. However, only a few organic acids and phenolic acids showed difference between CCYV-infected and healthy cucumber plants. CONCLUSIONS: CCYV infection repressed the defensive flavonoids, terpeneoids metabolism but triggered the lipids, amino acids and nucleotides metabolism with the inoculation period. This result suggests that CCYV-infection makes cucumber plants more susceptible for whiteflies attack and CCYV infection. The reduction of defensive comounds and the increase of amino acids may be partially responsible for enhancing feeding preference of whiteflies to CCYV-infected hosts. CCYV may hijacked lipid metabolism for virus replication and assembly.

Impacts of Cucurbit Chlorotic Yellows Virus (CCYV) on Biological Characteristics of Its Vector Bemisia tabaci (Hemiptera: Aleyrodidae) MED Species.

Plant viruses can change the phenotypes and defense pathways of the host plants and the performance of their vectors to facilitate their transmission. Cucurbit chlorotic yellows virus (CCYV) (Crinivirus), a newly reported virus occurring on cucurbit plants and many other plant species, is transmitted specifically by Bemisia tabaci MEAM1 (B biotype) and MED (Q biotype) cryptic species in a semipersistent manner. This study evaluated the impacts of CCYV on B. tabaci to better understand the plant-virus-vector interactions. By using CCYV-B. tabaci MED-cucumber as the model, we investigated whether or how a semipersistent plant virus impacts the biology of its whitefly vector. CCYV mRNAs were detectable in nymphs from first to fourth instars and adults of B. tabaci with different titers. Nymph instar durations and adult longevity of female whiteflies greatly extended on CCYV-infected plants, but nymph instar durations and adult longevity of male whiteflies were not significantly influenced. In addition, the body length and oviposition increased in adults feeding on CCYV-infected plants, but the hatching rates of eggs and survival rates of different stages were not affected. Most interestingly, the sex ratio (male:female) significantly reduced to 0.5:1 in whitefly populations on CCYV-infected plants, while the ratio remained about 1:1 on healthy plants. These results indicated that CCYV can significantly impact the biological characteristics of its vector B. tabaci. It is speculated that CCYV and B. tabaci have established a typical mutualist relationship mediated by host plants.

Identification and Physicochemical Properties of the Novel Hemolysin(s) From Oral Secretions of Helicoverpa armigera (Lepidoptera: Noctuidae).

Hemolysins cause the lysis of invading organisms, representing major humoral immunity used by invertebrates. Hemolysins have been discovered in hemolymph of Helicoverpa armigera larvae as immune factors. As oral immunity is great important to clear general pathogens, we presumed that hemolysins may be present in oral secretions (OS). To confirm this hypothesis, we conducted four testing methods to identify hemolysin(s) in larval OS of H. armigera, and analyzed physicochemical properties of the hemolysin in comparison with hemolytic melittin of Apis mellifera (L.) (Hymenoptera: Apidae) venom. We found hemolysin(s) from OS of H. armigera for the first time, and further identified in other lepidopteran herbivores. It could be precipitated by ammonium sulfate, which demonstrates that the hemolytic factor is proteinaceous. Labial gland showed significantly higher hemolytic activity than gut tissues, suggesting that hemolysin of OS is mainly derived from saliva secreted by labial glands. Physicochemical properties of hemolysin in caterpillar's OS were different from bee venom. It was noteworthy that hemolytic activity of OS was only partially inhibited even at 100°C. Hemolytic activity of OS was not inhibited by nine tested carbohydrates contrary to bee venom melittin. Moreover, effects of metal ions on hemolytic activity were different between OS and bee venom. We conclude that there is at least a novel hemolysin in OS of herbivorous insects with proposed antibacterial function, and its hemolytic mechanism may be different from melittin. Our study enriches understanding of the potential role of hemolysins in insect immunity and provides useful data to the field of herbivorous insect-pathogen research.

Changes in Bemisia tabaci feeding behaviors caused directly and indirectly by cucurbit chlorotic yellows virus.

BACKGROUND: Plant viruses can affect vector's behaviors in order to enhance viral transmission. Cucurbit chlorotic yellows virus (CCYV) (genus Crinivirus) is an emergent RNA plant virus and is transmitted specifically by biotypes B and Q of tobacco whitefly, Bemisia tabaci (Gennadius), in a semipersistent manner. METHODS: We used the electrical penetration graph (EPG) to investigate the effect of CCYV on the feeding behaviors of B. tabaci biotypes B and Q. RESULTS: CCYV could affect, both directly and indirectly, the feeding behaviors of B. tabaci to various degrees, depending on biotypes and sexes of the insect. CCYV showed stronger direct effects on biotype Q than on biotype B in terms of increased non-phloem probing and phloem salivation. CCYV increased non-phloem probing and phloem salivation more on females than on males of biotype Q, and increased phloem salivation more on females than on males of biotype B. CCYV had stronger indirect effects, via virus-infested plants, on biotype B than on biotype Q by enhancing phloem sap ingestion and feeding bouts. CCYV increased non-phloem probing and feeding bouts more on males than on females of biotype B, and decreased phloem sap ingestion more on males than on females on biotype Q indirectly. CONCLUSIONS: The results clearly indicated that CCYV affects the feeding behaviors of B. tabaci, which may lead to increased ability of the B. tabaci for CCYV transmission.

Infectious clones of the crinivirus cucurbit chlorotic yellows virus are competent for plant systemic infection and vector transmission.

Cucurbit chlorotic yellows virus (CCYV), a recently identified bipartite crinivirus, causes economic losses in cucurbit plants. CCYV is naturally transmitted only by whitefly Bemisia tabaci. Here we constructed full-length cDNA clones of CCYV (RNA1 and RNA2) fused to the T7 RNA polymerase promoter and the cauliflower mosaic virus 35S promoter. CCYV replicated and accumulated efficiently in Cucumis sativus protoplasts transfected with in vitro transcripts. Without RNA2, RNA1 replicated efficiently in C. sativus protoplasts. Agroinoculation with the infectious cDNA clones of CCYV resulted in systemic infection in the host plants of C. sativus and Nicotiana benthamiana. Virus derived from the infectious clones could be transmitted between cucumber plants by vector whiteflies. This system will greatly enhance the reverse genetic studies of CCYV gene functions.

Molecular Mechanism Underlying ROS-Mediated AKH Resistance to Imidacloprid in Whitefly.

Synthetic insecticides used to control Bemisia tabaci include organophosphorus, pyrethroids, insect growth regulators, nicotinoids, and neonicotinoids. Among these, neonicotinoids have been used continuously, which has led to the emergence of high-level resistance to this class of chemical insecticides in the whitefly, making whitefly management difficult. The adipokinetic hormone gene (AKH) and reactive oxygen species (ROS) play roles in the development of insect resistance. Therefore, the roles of AKH and ROS in imidacloprid resistance in Bemisia tabaci Mediterranean (MED; formerly biotype Q) were evaluated in this study. The expression level of AKH in resistant B. tabaci MED was significantly lower than that in sensitive B. tabaci (MED) (p < 0.05). AKH expression showed a decreasing trend. After AKH silencing by RNAi, we found that ROS levels as well as the expression levels of the resistance gene CYP6CM1 and its upstream regulatory factors CREB, ERK, and P38 increased significantly (p < 0.05); additionally, whitefly resistance to imidacloprid increased and mortality decreased (p < 0.001). These results suggest that AKH regulates the expression of resistance genes via ROS in Bemisia tabaci.

Managing Super Pests: Interplay between Pathogens and Symbionts Informs Biocontrol of Whiteflies.

Bemisia tabaci is distributed globally and incurs considerable economic and ecological costs as an agricultural pest and viral vector. The entomopathogenic fungus Metarhizium anisopliae has been known for its insecticidal activity, but its impacts on whiteflies are understudied. We investigated how infection with the semi-persistently transmitted Cucurbit chlorotic yellows virus (CCYV) affects whitefly susceptibility to M. anisopliae exposure. We discovered that viruliferous whiteflies exhibited increased mortality when fungus infection was present compared to non-viruliferous insects. High throughput 16S rRNA sequencing also revealed significant alterations of the whitefly bacterial microbiome diversity and structure due to both CCYV and fungal presence. Specifically, the obligate symbiont Portiera decreased in relative abundance in viruliferous whiteflies exposed to M. anisopliae. Facultative Hamiltonella and Rickettsia symbionts exhibited variability across groups but dominated in fungus-treated non-viruliferous whiteflies. Our results illuminate triangular interplay between pest insects, their pathogens, and symbionts-dynamics which can inform integrated management strategies leveraging biopesticides This work underscores the promise of M. anisopliae for sustainable whitefly control while laying the groundwork for elucidating mechanisms behind microbe-mediated shifts in vector competence.

Fine structure and primary sensory projections of sensilla located in the labial-palp pit organ of Helicoverpa armigera (Insecta).

The fine structure and primary sensory projections of sensilla located in the labial-palp pit organ of the cotton bollworm Helicoverpa armigera (Insecta, Lepidoptera) are investigated by scanning electron and transmission electron microscopy combined with confocal laser scanning microscopy. The pit organ located on the third segment of the labial palp is about 300 µm deep with a 60-µm-wide opening, each structure containing about 1200 sensilla. Two sensillum types have been found, namely hair-shaped and club-shaped sensilla, located on the upper and lower half of the pit, respectively. Most sensilla possess a single dendrite. The dendrite housed by the club-shaped sensilla is often split into several branches or becomes lamellated in the outer segment. As reported previously, the sensory axons of the sensilla in the labial pit organ form a bundle entering the ipsilateral side of the subesophageal ganglion via the labial palp nerve and project to three distinct areas: the labial pit organ glomerulus in each antennal lobe, the subesophageal ganglion and the ventral nerve cord. In the antennal lobe, the labial pit organ glomerulus is innervated by sensory axons from the labial pit organ only; no antennal afferents target this unit. One neuron has been found extending fine processes into the subesophageal ganglion and innervating the labial palp via one branch passing at the base of the labial palp nerve. The soma of this assumed motor neuron is located in the ipsilateral cell body layer of the subesophageal ganglion. Our results provide valuable knowledge concerning the neural circuit encoding information about carbon dioxide and should stimulate further investigations directed at controlling pest species such as H. armigera.

A plant virus enhances odorant-binding protein 5 (OBP5) in the vector whitefly for more actively olfactory orientation to the host plant.

BACKGROUND: The whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) is a notorious agricultural pest and the effective vector of many plant viruses worldwide. Cucurbit chlorotic yellows virus (CCYV), exclusively transmitted by B. tabaci in a semipersistent manner, is a serious causal agent in cucurbit crops in many countries. Plant viruses can manipulate the behaviors of insect vectors to promote the spread of themselves, but underlying mechanisms are remaining unclear. RESULTS: In this study, our observations indicated that B. tabaci, when carrying CCYV, oriented more actively to the host plant cucumber. Transcriptome analysis and quantitative polymerase chain reaction with reverse transcription analysis showed that the odorant-binding protein 5 (OBP5) was upregulated with viral acquisition. Sequence and phylogenetic analysis showed that BtabOBP5 was highly homologous with nine OBPs from other hemipteran insects. In addition, OBP5-silenced whiteflies significantly altered their orientation behavior towards cucumber plants and towards some typical volatile organic compounds released from cucumbers. CONCLUSION: This study described a novel mechanism by which the olfactory system of vector insects could be regulated by a semipersistent plant virus, thereby affecting insect olfactory behavior and relationship with host plants. These results provided a basis for developing potential olfaction-based pest management strategies in the future. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Discovery of novel whitefly vector proteins that interact with a virus capsid component mediating virion retention and transmission.

Specificity and efficiency of plant virus transmission depend largely on protein-protein interactions of vectors and viruses. Cucurbit chlorotic yellows virus (CCYV), transmitted specifically by tobacco whitefly, Bemisia tabaci, in a semi-persistent manner, has caused serious damage on cucurbit and vegetable crops around the world. However, the molecular mechanism of interaction during CCYV retention and transmission are still lacking. CCYV was proven to bind particularly to the whitefly foregut, and here, we confirmed that the minor coat protein (CPm) of CCYV is participated in the interaction with the vector. In order to identify proteins of B. tabaci that interact directly with CPm of CCYV, the immunoprecipitation (IP) assay and DUALmembrane cDNA library screening technology were applied. The cytochrome c oxidase subunit 5A (COX), tubulin beta chain (TUB) and keratin, type I cytoskeletal 9-like (KRT) of B. tabaci shown strong interactions with CPm and are closely associated with the retention within the vector and transmission of CCYV. These findings on whitefly protein-CCYV CPm interactions are crucial for a much better understanding the mechanism of semi-persistent plant virus transmission by insect vectors, as well as for implement new strategies for effective management of plant viruses and their vector insects.

Transcriptomics Reveals the Killing Mechanism by Which Entomopathogenic Fungi Manipulate the RNA Expression Profiles of Termites and Provides Inspiration for Green Pest Management.

As chemical pesticides have caused serious environmental pollution, fungus-based biological control has become a developing alternative to chemical control. Here, we aimed to determine the molecular mechanism underlying how Metarhizium anisopliae facilitated invasive infection. We found that the fungus increased its virulence by downregulating glutathione S-transferase (GST) and superoxide dismutase (SOD) throughout termite bodies. Among 13 fungus-induced microRNAs throughout termite bodies, miR-7885-5p and miR-252b upregulation significantly downregulated several mRNAs in response to toxic substances to increase the fungal virulence [e.g., phosphoenolpyruvate carboxykinase (GTP) and heat shock protein homologue SSE1]. In addition, nanodelivered small interfering RNA of GST and SOD and miR-7885-5p and miR-252b mimics increased the virulence of the fungus. These findings provide new insights into the killing mechanism of entomopathogens and their utilization of the host miRNA machinery to reduce host defenses, laying the groundwork to enhance virulence of biocontrol agents for green pest management.

Complete Mitochondrial Genome of Malenka flexura (Plecoptera: Nemouridae) and Phylogenetic Analysis.

The genus-level relationships within the subfamily Amphinemurinae have been controversial, although attempts have been made based on morphology and limited molecular data. With the establishment of two new genera, the phylogenetic relationships within Amphinemurinae should be re-examined. In this study, the complete mitochondrial genome (mitogenome) of Malenka flexura of the genus Malenka was firstly sequenced and analyzed. The phylogeny of Amphinemurinae was also reconstructed using 13 proteincoding genes (PCGs) from previously published stoneflies. This mitogenome was 15,744 bp long and encoded the typical 37 genes, as well as a putative control region. The gene arrangement of M. flexura mitogenome is identical with the putative ancestral mitogenome in Drosophila yakuba. Most PCGs used standard ATN as start codons and TAA/TAG as termination codons. All tRNA genes exhibited the typical cloverleaf secondary structure, except for tRNASer(AGN), whose dihydrouridine (DHU) arm was lacking. Some structural elements in the control region were founded, such as tandem repeat regions, stemloop structures, polyN stretch and microsatellite structure, etc. Phylogenetic analyses of sequenced Amphinemurinae mitogenomes unsupported the sister relationship of Amphinemura and Malenka. Finally, the phylogenetic analyses inferred a relationship within Amphinemurinae: Amphinemura + (Malenka + (Protonemura + (Indonemoura + (Sphaeronemoura + Mesonemoura)))).

Influence of Oilseed Rape Seed Treatment with Imidacloprid on Survival, Feeding Behavior, and Detoxifying Enzymes of Mustard Aphid, Lipaphis erysimi.

Imidacloprid application, as a seed coating agent on oilseed rape, is recommended to control mustard aphid, Lipaphis erysimi (Kaltenbach) (Hemiptera: Aphididae). In this study, responses of L. erysimi were investigated, including survival, feeding behavior, and detoxifying enzymes, on the oilseed rape seedlings grown from seeds coated with imidacloprid at rates of 6, 12, or 18 g active ingredient (a.i.)/kg seed. The results showed that the aphids' survival rate, together with that of the progeny of the survivors, on the seed-treated seedlings significantly decreased. This indicates that the aphid population in fields can be suppressed effectively. The electrical penetration graph (EPG) technique was used to record aphid feeding behaviors on two-, four-, and six-leaf stages of oilseed rape seedlings that had been seed-coated with imidacloprid, and individual responses were revealed during the aphid feeding behavior. On the plants at the two-leaf stage, aphid feeding behaviors were influenced, showing decreased frequency of stylet penetration into the leaf (probe) or into the mesophyll cells (potential drops, pds for short), and shortened duration of stylet event in the leaf (probe) or in the phloem. On the plants at the four- and six-leaf stages, these impacts of imidacloprid were weakened; however, the saliva secretion duration in phloem was shortened to less than 5 min in all imidacloprid treatments. The activity of mixed-function oxidase in aphids maintained on the treated seedlings with imidacloprid was elevated. In conclusion, imidacloprid could be used as a seed coating agent for aphid control, but chemical resistance in aphids should not be ignored.

Leaf surface factors of transgenic Bt cotton associated with the feeding behaviors of cotton aphids: a case study on non-target effects.

The present paper reports case study results of the risk assessment of transgenic Bt cotton on a non-target pest, cotton aphid, Aphis gossypii. Several types of techniques, i.e., electrical penetration graph (EPG), light and electron microscopy, bioassays and chemical analysis, were applied to investigate physical and chemical leaf factors of 2 transgenic Bt cotton lines (GK12 and GK19) and their parental non-Bt cotton line (Simian3) associated with searching and feeding behaviors of cotton aphids on leaves or leaf extracts of cotton plants. EPG results showed that there were some differences among behaviors of cotton aphids on 2 Bt cotton and 1 non-Bt cotton lines. Cotton aphids performed similarly to leaf surface extracts from 3 cotton lines; and leaf surface chemicals, mainly volatiles and waxes, were almost identical in the components and concentrations among the cotton lines. However, three cotton lines were quite different from each other in the densities of certain kinds of covering trichomes. Therefore, the relationships between the physical characteristics and the searching behaviors of cotton aphids on the three cotton lines were constructed as the regression equations. Glandular trichomes and covering trichomes with 5 branches influenced the cotton aphids' searching behaviors effectively; and other trichomes with other branches affected aphids in varying ways. These results demonstrated that leaf surface physical factors of transgenic Bt cotton lines different from their parental non-Bt line could affect the penetration behaviors of non-target cotton aphids. Cotton aphids penetrate and feed more easily on two Bt cotton lines than on the non-Bt cotton line.

Microsatellites for Phytolacca acinosa (Phytolaccaceae), a traditional medicinal herb.

PREMISE OF THE STUDY: Phytolacca acinosa (Phytolaccaceae) is a traditional Chinese herb with multiple medicinal uses and is an important pigment source. Due to excessive human harvesting, the population numbers and sizes have decreased dramatically. METHODS AND RESULTS: Using an enriched genomic library, we developed and characterized 15 microsatellite primers for P. acinosa, 13 of which were polymorphic. The number of alleles varied from two to seven. The observed heterozygosity and expected heterozygosity per locus ranged from 0.267 to 1.000 and 0.331 to 0.743, respectively. All of the primers that were developed were also successfully applied in P. americana. CONCLUSIONS: These markers should be useful in probing the genetic diversity, genetic structure, and mating systems of P. acinosa, which could provide information about protecting and sustainably harvesting this species.

A Semipersistent Plant Virus Differentially Manipulates Feeding Behaviors of Different Sexes and Biotypes of Its Whitefly Vector.

It is known that plant viruses can change the performance of their vectors. However, there have been no reports on whether or how a semipersistent plant virus manipulates the feeding behaviors of its whitefly vectors. Cucurbit chlorotic yellows virus (CCYV) (genus Crinivirus, family Closteroviridae) is an emergent plant virus in many Asian countries and is transmitted specifically by B and Q biotypes of tobacco whitefly, Bemisia tabaci (Gennadius), in a semipersistent manner. In the present study, we used electrical penetration graph (EPG) technique to investigate the effect of CCYV on the feeding behaviors of B. tabaci. The results showed that CCYV altered feeding behaviors of both biotypes and sexes of B. tabaci with different degrees. CCYV had stronger effects on feeding behaviors of Q biotype than those of B biotype, by increasing duration of phloem salivation and sap ingestion, and could differentially manipulate feeding behaviors of males and females in both biotype whiteflies, with more phloem ingestion in Q biotype males and more non-phloem probing in B biotype males than their respective females. With regard to feeding behaviors related to virus transmission, these results indicated that, when carrying CCYV, B. tabaci Q biotype plays more roles than B biotype, and males make greater contribution than females.

Genome sequencing of the sweetpotato whitefly Bemisia tabaci MED/Q.

The sweetpotato whitefly Bemisia tabaci is a highly destructive agricultural and ornamental crop pest. It damages host plants through both phloem feeding and vectoring plant pathogens. Introductions of B. tabaci are difficult to quarantine and eradicate because of its high reproductive rates, broad host plant range, and insecticide resistance. A total of 791 Gb of raw DNA sequence from whole genome shotgun sequencing, and 13 BAC pooling libraries were generated by Illumina sequencing using different combinations of mate-pair and pair-end libraries. Assembly gave a final genome with a scaffold N50 of 437 kb, and a total length of 658 Mb. Annotation of repetitive elements and coding regions resulted in 265.0 Mb TEs (40.3%) and 20 786 protein-coding genes with putative gene family expansions, respectively. Phylogenetic analysis based on orthologs across 14 arthropod taxa suggested that MED/Q is clustered into a hemipteran clade containing A. pisum and is a sister lineage to a clade containing both R. prolixus and N. lugens. Genome completeness, as estimated using the CEGMA and Benchmarking Universal Single-Copy Orthologs pipelines, reached 96% and 79%. These MED/Q genomic resources lay a foundation for future 'pan-genomic' comparisons of invasive vs. noninvasive, invasive vs. invasive, and native vs. exotic Bemisia, which, in return, will open up new avenues of investigation into whitefly biology, evolution, and management.

Correlation of stylet activities by the glassy-winged sharpshooter, Homalodisca coagulata (Say), with electrical penetration graph (EPG) waveforms.

Glassy-winged sharpshooter, Homalodisca coagulata (Say), is an efficient vector of Xylella fastidiosa (Xf), the causal bacterium of Pierce's disease, and leaf scorch in almond and oleander. Acquisition and inoculation of Xf occur sometime during the process of stylet penetration into the plant. That process is most rigorously studied via electrical penetration graph (EPG) monitoring of insect feeding. This study provides part of the crucial biological meanings that define the waveforms of each new insect species recorded by EPG. By synchronizing AC EPG waveforms with high-magnification video of H. coagulata stylet penetration in artifical diet, we correlated stylet activities with three previously described EPG pathway waveforms, A1, B1 and B2, as well as one ingestion waveform, C. Waveform A1 occured at the beginning of stylet penetration. This waveform was correlated with salivary sheath trunk formation, repetitive stylet movements involving retraction of both maxillary stylets and one mandibular stylet, extension of the stylet fascicle, and the fluttering-like movements of the maxillary stylet tips. Waveform B1 was ubitquious, interspersed throughout the other waveforms. B1 sub-type B1w was correlated with salivation followed by maxillary tip fluttering. This tip fluttering also occurred before and during B1 sub-type B1s, but was not directly correlated with either the occurrence or frequency of this waveform. Waveform B2 was correlated with sawing-like maxillary stylet movements, which usually occurred during salivary sheath branching. Waveform C was correlated with ingestion. Fluid outflow was also observed as a mechanism to clear the maxillary tips from debris during waveform C. This detailed understanding of stylet penetration behaviors of H. coagulata is an important step toward identifying the instant of bacterial inoculation which, in turn, will be applied to studies of disease epidemiology and development of host plant resistance.

Distribution and variations of three 1,4-benzoxazin-3-ones in maize induced by the Asian corn borer, Ostrinia furnacalis (Guenée).

Contents of three 1,4-benzoxazin-3-ones in tissue samples from different parts (young leaf, second leaf, old leaf, stem and root) of young maize plants of 4-leaves stage, fed by the third instar larvae of the Asian corn borer, Ostrinia furnacalis (Guenée), were analyzed by high-performance liquid chromatography-mass spectroscopy (HPLC-MS). Samples were taken immediately (set A) or 48 h (set B) after larvae had fed on the second leaf for 48 h. The three 1,4-benzoxazin-3-ones investigated in our experiments were 2,4-dihydroxy-7-methoxy-1,4(2H)-benzoxazin-3-one (DIMBOA), 2,4-dihydroxy-1,4(2H)-benzoxazin-3-one (DIBOA) and 2-hydroxy-7-methoxy-1,4(2H)-benzoxazin-3-one (HMBOA). In samples of set A, the levels of DIMBOA and HMBOA were significantly lifted in the old leaf (L3) and young leaf (L1), respectively, while amounts of these two chemicals in other plant parts were not significantly different between larvae-fed plants and intact plants. Concentrations of DIBOA in each plant part remained unchanged. In samples of set B, no concentration differences for any of these three 1,4-benzoxazin-3-ones between larvae-fed plants and controls were observed in any plant part. The feeding of the Asian corn borer seems to have limited effects on induction of these three 1,4-benzoxazin-3-ones in young maize plants of the variety investigated.

Phylogenetic relationships of Hemiptera inferred from mitochondrial and nuclear genes.

Here, we reconstructed the Hemiptera phylogeny based on the expanded mitochondrial protein-coding genes and the nuclear 18S rRNA gene, separately. The differential rates of change across lineages may associate with long-branch attraction (LBA) effect and result in conflicting estimates of phylogeny from different types of data. To reduce the potential effects of systematic biases on inferences of topology, various data coding schemes, site removal method, and different algorithms were utilized in phylogenetic reconstruction. We show that the outgroups Phthiraptera, Thysanoptera, and the ingroup Sternorrhyncha share similar base composition, and exhibit "long branches" relative to other hemipterans. Thus, the long-branch attraction between these groups is suspected to cause the failure of recovering Hemiptera under the homogeneous model. In contrast, a monophyletic Hemiptera is supported when heterogeneous model is utilized in the analysis. Although higher level phylogenetic relationships within Hemiptera remain to be answered, consensus between analyses is beginning to converge on a stable phylogeny.