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Deciphering cell cycle phases of polyphenic tissues is an important challenge in understanding the cellular mechanism of polymorphism. We use flow cytometry to analyze cell cycle phases of short wings and long wings of the brown planthopper. This provides information on the arresting cell cycle phases in different wing forms. The protocol could be applied to analysis of the cell cycle phases of other polyphenic insects and in different polyphenic tissues after modification. For complete details on the use and execution of this protocol, please refer to Lin et al. (2020)., Competing Interests: The authors declare no competing interests., (© 2020 The Authors.)

Certain families of plant-feeding insects in the order Hemiptera (infraorder Pentatomomorpha) have established symbiotic relationships with microbes that inhabit specific pouches (caeca) of their midgut epithelium. The placement of these caeca in a well-delineated region at the most posterior end of the midgut bordering the hindgut is conserved in these families; in situ the convoluted midgut is predictably folded so that this caecal region lies adjacent to the anterior-most region of the midgut. Depending on the hemipteran family, caeca vary in their number and configuration at a given anterior-posterior location. At the host-microbe interface, epithelial plasma membranes of midgut epithelial cells interact with nonself antigens of microbial surfaces. In the different hemipteran species examined, a continuum of interactions is observed between microbes and host membranes. Bacteria can exist as free living cells within the midgut lumen without contacting host membranes while other host cells physically interact extensively with microbial surfaces by extending numerous processes that interdigitate with microbes; and, in many instances, processes completely envelope the microbes. The host cells can embrace the foreign microbes, completely enveloping each with a single host membrane or sometimes enveloping each with the two additional host membranes of a phagosome., (© 2019 Wiley Periodicals, Inc.)

Background: Whiteflies (Bemisia tabaci) are phloem sap-sucking pests that because of their broad host range and ability to transmit viruses damage crop plants worldwide. B. tabaci are now known to be a complex of cryptic species that differ from each other in many characteristics such as mode of interaction with viruses, invasiveness, and resistance to insecticides. Asia II 1 is an indigenous species found on the Indian sub-continent and south-east Asia while the species named as Middle East Asia Minor 1 (MEAM1), likely originated from the Middle-East and has spread worldwide in recent decades. The purpose of this study is to find genomic differences between these two species., Results: Sequencing of the nuclear genome of Asia II 1 with Illumina HiSeq and MiSeq generated 198.90 million reads that covers 88% of the reference genome. The sequence comparison with MEAM1 identified 2,327,972 SNPs and 202,479 INDELs. In Total, 1294 genes were detected with high impact variants. The functional analysis revealed that some of the genes are involved in virus transmission including 4 genes in Tomato yellow leaf curl virus (TYLCV) transmission, 96 in Tomato crinivirus (ToCV) transmission, and 14 genes in insecticide resistance., Conclusions: These genetic differences between Asia II 1 and MEAM1 may underlie the major biological differences between the two species such as virus transmission, insecticide resistance, and range of host plants. The present study provides new genomic data and information resources for Asia II 1 that will not only contribute to the species delimitation of whitefly, but also help in conceiving future research studies to develop more targeted management strategies against whitefly.

To visualize phytoplasmas at early stages of vector infection, an immunofluorescence assay was developed. The chapter provides experimental details on dissection of salivary glands, incubation of the dissected organs with phytoplasma suspension, fixation, embedding, sectioning, labeling, and final visualization with confocal microscopy. All the procedure will be described for the leafhopper Euscelidius variegatus, natural vector of "Candidatus phytoplasma asteris" and laboratory vector of Flavescence dorée phytoplasma.

Plant sap-feeding insects (Hemiptera) rely on bacterial symbionts for nutrition absent in their diets. These bacteria experience extreme genome reduction and require genetic resources from their hosts, particularly for basic cellular processes other than nutrition synthesis. The host-derived mechanisms that complete these processes have remained poorly understood. It is also unclear how hosts meet the distinct needs of multiple bacterial partners with differentially degraded genomes. To address these questions, we investigated the cell-specific gene-expression patterns in the symbiotic organs of the aster leafhopper (ALF), Macrosteles quadrilineatus (Cicadellidae). ALF harbors two intracellular symbionts that have two of the smallest known bacterial genomes: Nasuia (112 kb) and Sulcia (190 kb). Symbionts are segregated into distinct host cell types (bacteriocytes) and vary widely in their basic cellular capabilities. ALF differentially expresses thousands of genes between the bacteriocyte types to meet the functional needs of each symbiont, including the provisioning of metabolites and support of cellular processes. For example, the host highly expresses genes in the bacteriocytes that likely complement gene losses in nucleic acid synthesis, DNA repair mechanisms, transcription, and translation. Such genes are required to function in the bacterial cytosol. Many host genes comprising these support mechanisms are derived from the evolution of novel functional traits via horizontally transferred genes, reassigned mitochondrial support genes, and gene duplications with bacteriocyte-specific expression. Comparison across other hemipteran lineages reveals that hosts generally support the incomplete symbiont cellular processes, but the origins of these support mechanisms are generally specific to the host-symbiont system., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Background: In China, the rice pathogen Rice yellow stunt virus (RYSV), a member of the genus Nucleorhabdovirus in the family Rhabdoviridae, was a severe threat to rice production during the1960s and1970s. Fundamental aspects of the biology of this virus such as protein localization and formation of the RYSV viroplasm during infection of insect vector cells are largely unexplored. The specific role(s) of the structural proteins nucleoprotein (N) and phosphoprotein (P) in the assembly of the viroplasm during RYSV infection in insect vector is also unclear., Methods: In present study, we used continuous leafhopper cell culture, immunocytochemical techniques, and transmission electron microscopy to investigate the subcellular distributions of N and P during RYSV infection. Both GST pull-down assay and yeast two-hybrid assay were used to assess the in vitro interaction of N and P. The dsRNA interference assay was performed to study the functional roles of N and P in the assembly of RYSV viroplasm., Results: Here we demonstrated that N and P colocalized in the nucleus of RYSV-infected Nephotettix cincticeps cell and formed viroplasm-like structures (VpLSs). The transiently expressed N and P are sufficient to form VpLSs in the Sf9 cells. In addition, the interactions of N/P, N/N and P/P were confirmed in vitro. More interestingly, the accumulation of RYSV was significantly reduced when the transcription of N gene or P gene was knocked down by dsRNA treatment., Conclusions: In summary, our results suggest that N and P are the main viral factors responsible for the formation of viroplasm in RYSV-infected insect cells. Early during RYSV infection in the insect vector, N and P interacted with each other in the nucleus to form viroplasm-like structures, which are essential for the infection of RYSV.

Temperature affects the persistence of diverse symbionts of insects. Our previous study indicates that the whitefly symbionts confined within bacteriocytes or scattered throughout the body cavity outside bacteriocytes may have differential thermal sensitivity. However, the underlying mechanisms remain largely unknown. Here, we report that following continuous heat stress, Portiera and Hamiltonella were almost completely depleted in two species of Middle East-Asia Minor 1 (MEAM1) and Mediterranean (MED) of the Bemisia tabaci whitefly cryptic species complex. Meanwhile, proliferation of bacteriocytes was severely inhibited and approximately 50% of the nymphs had lost one of the two bacteriomes. While cell size of bacteriocytes was increased, cell number was severely decreased leading to reduction of total volume of bacteriocytes. Moreover, bacteriocyte organelles and associated symbionts were lysed, and huge amount of electron-dense inclusions accumulated. Eventually, Portiera and Hamiltonella failed to be transmitted to the next generation. In contrast, Rickettsia could be detected although at a reduced level, and successfully transmitted to eggs. The results suggest that the thermal sensitivity of bacteriocytes may limit thermal tolerance and vertical transmission of the associated symbionts, and consequently different patterns of distribution of symbionts may affect their capacity to tolerate unfavourable temperatures and persistence in the host., (© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.)

In meiosis I, homologous chromosomes combine to form bivalents, which align on the metaphase plate. Homologous chromosomes then separate in anaphase I. Univalent sex chromosomes, on the other hand, are unable to segregate in the same way as homologous chromosomes of bivalents due to their lack of a homologous pairing partner in meiosis I. Here, we studied univalent segregation in a Hemipteran insect: the spittlebug Philaenus spumarius. We determined the chromosome number and sex determination mechanism in our population of P. spumarius and showed that, in male meiosis I, there is a univalent X chromosome. We discovered that the univalent X chromosome in primary spermatocytes forms an amphitelic attachment to the spindle and aligns on the metaphase plate with the autosomes. Interestingly, the X chromosome remains at spindle midzone long after the autosomes have separated. In late anaphase I, the X chromosome initiates movement towards one spindle pole. This movement appears to be correlated with a loss of microtubule connections between the kinetochore of one chromatid and its associated spindle pole.

In this study, by using light and electron microscopy, we describe the sperm morphology of the leafhopper Diaphorina citri, a serious pest of citrus throughout the world. In this species the sperm measures 538.49±8.75μm in length, and as observed in psylloids, the sperm, when manipulated, opens into two filaments, one of which is attached to the nuclear base and the other becomes free. Along the flagellum, and only of it, there are lateral projections, about 2μm in length. Furthermore, at the end of the flagellum three appendages, with approximately 7μm in length, are observed. The head region is formed by the nucleus with compact chromatin, and, parallel to it, a structure of median electron density that extends about 25μm in length ahead of the nuclear tip. The flagellum consists of an axoneme with a 9+9+2 microtubule arrangement, two mitochondrial derivatives, and two accessory bodies each with two regions of different electron density. The presence of lateral projections is a characteristic observed in other Sternorrhyncha. As seen previously, the sperm opening in two filaments, when manipulated, was observed only in Psylloidea, and the presence of the three appendices at the end of the flagellum distinguishes D. citri from the other Psylloidea species studied., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Prostaglandins (PGs) are oxygenated metabolites of arachidonic acid (AA) and two other C20 polyunsaturated fatty acids that serve as biochemical signals mediating physiological functions. We reported that PGs influence protein expression in insect cell lines, which prompted the question: do PGs influence cell proliferation or viability in insect cell lines? Here, we report on the outcomes of experiments designed to address the question in cell lines from three insect orders: Hemiptera (squash bug, Anasa tristis, BCIRL-AtE-CLG15A), Coleoptera (red flour beetle, Tribolium castaneum, BCIRL-TcA-CLG1), and Lepidoptera (tobacco budworm, Heliothis virescens, BCIRL-HvAM1). Treating the insect cell lines with PGA 1 , PGA 2 , or PGD 2 led to dose-dependent reductions in cell numbers. All three cell lines were sensitive to PGA 1 and PGA 2 (IC 50 s = 9.9 to 26.9 μM) and were less sensitive to PGD 2 (IC 50 s = 31.6 to 104.7 μM). PG treatments also led to cell death at higher concentrations, as seen in mammalian cell lines. PGE 1 , PGE 2 , and PGF 2α treatments did not influence AtE-CLG15A or HvAM1 cell numbers at lower concentrations, but led to dose-related reductions in TcA-CLG1 cells at higher concentrations. Similar treatments with pharmaceutical inhibitors of PG biosynthesis also led to reduced cell numbers: MAFP (inhibits phospholipase A 2 ), indomethacin (inhibits PG biosynthesis), and esculetin (inhibits lipoxygenase). Because these pharmaceuticals are used to relieve inflammation and other medical issues in human medicine, they are not toxic to animal cells. We infer PGs are necessary in optimal quantities for ongoing homeostatic functions in established cell lines; in quantities outside the optimal concentrations, PGs are deleterious.

The squash bug, Anasa tristis, is a pest of cucurbits that exerts direct damage on crops and is a vector of plant pathogens. We established cell lines from this insect to serve as tools for basic biology, including virology and immunology, as well as applied studies, such as insecticide development programs. We initiated 15 cell cultures, using nine media or combinations of media. The media yielding the best results were a modification of Kimura's medium and a combination of two commercially available cell culture media (EX-CELL 420 and L15). We designated the two cell lines as BCIRL-AtE-CLG11 and BCIRL-AtE-CLG15. From the AtE-CLG15 line, we isolated two sub-lines, A and B. Of these, the most consistently replicating line was AtE-CLG15A. We determined the doubling time of this line (190 h) and its mean cell diameter (14.5 ± 0.7 μm). We characterized the AtE-CLG15A line using DAF-PCR. The BCIRL-AtE-CLG15A cell line is now available for researchers world-wide.

Candidatus Liberibacter asiaticus (CLas) is a phloem-limited, gram-negative, fastidious bacterium that is associated with the development of citrus greening disease, also known as Huanglongbing (HLB). CLas is transmitted by the Asian citrus psyllid (ACP) Diaphorina citri, in a circulative manner. Two major barriers to transmission within the insect are the midgut and the salivary glands. We performed a thorough microscopic analysis within the insect midgut following exposure to CLas-infected citrus trees. We observed changes in nuclear architecture, including pyknosis and karyorrhexis as well as changes to the actin cytoskeleton in CLas-exposed midgut cells. Further analyses showed that the changes are likely due to the activation of programmed cell death as assessed by Annexin V staining and DNA fragmentation assays. These results suggest that exposure to CLas-infected trees induces apoptotic responses in the psyllid midgut that should be further investigated. Understanding the adaptive significance of the apoptotic response has the potential to create new approaches for controlling HLB.

The development and organization of the ovaries of ten species from four Psylloidea families (Psyllidae, Triozidae, Aphalaridae and Liviidae) have been investigated. The ovaries of the last larval stage (i.e. fifth instar) of all examined species are filled with numerous clusters of cystocytes which undergo synchronous incomplete mitotic division. Cystocytes of the given cluster are arranged into a rosette with polyfusome in the centre. These clusters are associated with single somatic cells. At the end of the fifth instar, the clusters begin to separate from each other, forming spherical ovarioles which are surrounded by a single layer of somatic cells. In the ovarioles of very young females all cystocytes enter the prophase of meiosis and differentiate shortly thereafter into oocytes and trophocytes (nurse cells). Meanwhile, somatic cells differentiate into cells of the inner epithelial sheath surrounding the trophocytes and into the prefollicular cells that encompass the oocytes. During this final differentiation, the trophocytes lose their cell membranes and become syncytial. Oocytes remain cellular and most of them (termed arrested oocytes) do not grow. In the ovarioles of older females, one oocyte encompassed by its follicle cells starts growing, still connected to the syncytial tropharium by a nutritive cord. After the short phase of previtellogenesis alone, the oocyte enters its vitellogenic the growth phase in the vitellarium. At that time, the second oocyte may enter the vitellarium and start its previtellogenic growth. In the light of the obtained results, the phylogeny of psyllids, as well as phylogenetic relationships between taxa of Hemiptera: Sternorrhyncha are discussed., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The whitefly Bemisia tabaci MEAM1 species complex has invaded several parts of the world in the past 30 years and replaced native whitefly populations in the invaded regions, including certain areas of China. One of the possible reasons for the invasion is that MEAM1 whiteflies are more fecund than native species. However, the factors that affect the reproduction of the B. tabaci cryptic species are not clearly known. The regulation of oogenesis is thought to be one of the essential processes for egg formation and ovary development and could affect its population dynamics. In this study, the ovariole structure and oogenesis of the MEAM1 species complex was examined using light and transmission electron microscopy. Telotrophic ovarioles were observed in the MEAM1 species complex. Each ovariole had two well defined regions: the tropharium and the vitellarium. The tropharium always had more than ten trophocytes. The development of a single oocyte in the vitellarium has four phases: oocyte formation, previtellogenesis, vitellogenesis and choriogenesis. Two arrested oocytes, follicular cells and uncompleted oocytes were separated from the tropharium by microtubule and microfilaments. Early previtellogenesis oocytes absorbed nutrients and endosymbiont bacteria through a nutritive cord. However, the vitellogenesis of oocytes transmitted Vg through both the nutritive cord and the space between follicular cells. Each mature oocyte with deposited yolk proteins had only one bacteriocyte and was surrounded by a single layer of follicular cells. The oogenesis in the B. tabaci MEAM1 species complex concluded with the differentiation of oocytes, the transport of yolk and endosymbionts as well as the development and maturation of oocytes. This result provides important information that further defines the regulation of oogenesis in the B. tabaci complex., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Xylella fastidiosa is unique among insect-transmitted plant pathogens because it is propagative but noncirculative, adhering to and multiplying on the cuticular lining of the anterior foregut. Any inoculation mechanism for X. fastidiosa must explain how bacterial cells exit the vector's stylets via the food canal and directly enter the plant. A combined egestion-salivation mechanism has been proposed to explain these unique features. Egestion is the putative outward flow of fluid from the foregut via hypothesized bidirectional pumping of the cibarium. The present study traced green fluorescent protein-expressing X. fastidiosa or fluorescent nanoparticles acquired from artificial diets by glassy-winged sharpshooters, Homalodisca vitripennis, as they were egested into simultaneously secreted saliva. X. fastidiosa or nanoparticles were shown to mix with gelling saliva to form fluorescent deposits and salivary sheaths on artificial diets, providing the first direct, conclusive evidence of egestion by any hemipteran insect. Therefore, the present results strongly support an egestion-salivation mechanism of X. fastidiosa inoculation. Results also support that a column of fluid is transiently held in the foregut without being swallowed. Evidence also supports (but does not definitively prove) that bacteria were suspended in the column of fluid during the vector's transit from diet to diet, and were egested with the held fluid. Thus, we hypothesize that sharpshooters could be true "flying syringes," especially when inoculation occurs very soon after uptake of bacteria, suggesting the new paradigm of a nonpersistent X. fastidiosa transmission mechanism.

Background: Mutualistic obligate endosymbioses shape the evolution of endosymbiont genomes, but their impact on host genomes remains unclear. Insects of the sub-order Sternorrhyncha (Hemiptera) depend on bacterial endosymbionts for essential amino acids present at low abundances in their phloem-based diet. This obligate dependency has been proposed to explain why multiple amino acid transporter genes are maintained in the genomes of the insect hosts. We implemented phylogenetic comparative methods to test whether amino acid transporters have proliferated in sternorrhynchan genomes at rates grater than expected by chance., Results: By applying a series of methods to reconcile gene and species trees, inferring the size of gene families in ancestral lineages, and simulating the null process of birth and death in multi-gene families, we uncovered a 10-fold increase in duplication rate in the AAAP family of amino acid transporters within Sternorrhyncha. This gene family expansion was unmatched in other closely related clades lacking endosymbionts that provide essential amino acids., Conclusions: Our findings support the influence of obligate endosymbioses on host genome evolution by both inferring significant expansions of gene families involved in symbiotic interactions, and discovering increases in the rate of duplication associated with multiple emergences of obligate symbiosis in Sternorrhyncha.

The glassy-winged sharpshooter (Homalodisca vitripennis) is a highly vagile and polyphagous insect found throughout the southwestern United States. These insects are the predominant vectors of Xylella fastidiosa (X. fastidiosa), a xylem-limited bacterium that is the causal agent of Pierce's disease (PD) of grapevine. Pierce's disease is economically damaging; thus, H. vitripennis have become a target for pathogen management strategies. A dicistrovirus identified as Homalodisca coagulata virus-01 (HoCV-01) has been associated with an increased mortality in H. vitripennis populations. Because a host cell is required for HoCV-01 replication, cell culture provides a uniform environment for targeted replication that is logistically and economically valuable for biopesticide production. In this study, a system for large-scale propagation of H. vitripennis cells via tissue culture was developed, providing a viral replication mechanism. HoCV-01 was extracted from whole body insects and used to inoculate cultured H. vitripennis cells at varying levels. The culture medium was removed every 24 hr for 168 hr, RNA extracted and analyzed with qRT-PCR. Cells were stained with trypan blue and counted to quantify cell survivability using light microscopy. Whole virus particles were extracted up to 96 hr after infection, which was the time point determined to be before total cell culture collapse occurred. Cells were also subjected to fluorescent staining and viewed using confocal microscopy to investigate viral activity on F-actin attachment and nuclei integrity. The conclusion of this study is that H. vitripennis cells are capable of being cultured and used for mass production of HoCV-01 at a suitable level to allow production of a biopesticide.

Mutualisms that become evolutionarily stable give rise to organismal interdependencies. Some insects have developed intracellular associations with communities of bacteria, where the interdependencies are manifest in patterns of complementary gene loss and retention among members of the symbiosis. Here, using comparative genomics and microscopy, we show that a three-member symbiotic community has become a four-way assemblage through a novel bacterial lineage-splitting event. In some but not all cicada species of the genus Tettigades, the endosymbiont Candidatus Hodgkinia cicadicola has split into two new cytologically distinct but metabolically interdependent species. Although these new bacterial genomes are partitioned into discrete cell types, the intergenome patterns of gene loss and retention are almost perfectly complementary. These results defy easy classification: they show genomic patterns consistent with those observed after both speciation and whole-genome duplication. We suggest that our results highlight the potential power of nonadaptive forces in shaping organismal complexity., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Numerous virus pathogens are transmitted by specific arthropod vectors. Understanding the mechanism of transmission is a critical step in the epidemiology of plant viruses and is crucial for the development of effective disease control strategies. In this study, we describe the localization and distribution of Wheat dwarf virus (WDV), an economically important and widespread single-stranded DNA virus, in its leafhopper vector, Psammotettix alienus. The results suggest that WDV not only can move to the salivary glands from the anterior and middle midgut via the hemocoel but also can pass directly through the sheath of the filter chamber and be readily transmitted to healthy wheat plants within 5 min of an acquisition access period on infected plants. When a bacterial-expressed recombinant capsid protein (CP) was incubated with the internal organs of leafhoppers, CP-immunoreactive antigens were found at the anterior and middle midgut. Furthermore, when leafhoppers were fed with an antiserum raised against the CP, the accumulation of WDV in the gut cells, hemocoel, and salivary glands was significantly reduced. These data provide evidence that transmission of WDV is determined by a CP-mediated virion-vector retention mechanism.

Spiroplamas are helical, cell wall-less bacteria belonging to the Class Mollicutes, a group of microorganisms phylogenetically related to low G+C, Gram-positive bacteria. Spiroplasma species are all found associated with arthropods and a few, including Spiroplasma citri are pathogenic to plant. Thus S. citri has the ability to colonize cells of two very distinct hosts, the plant and the insect vector. While spiroplasmal factors involved in transmission by the leafhopper Circulifer haematoceps have been identified, their specific contribution to invasion of insect cells is poorly understood. In this study we provide evidence that the lipoprotein spiralin plays a major role in the very early step of cell invasion. Confocal laser scanning immunomicroscopy revealed a relocalization of spiralin at the contact zone of adhering spiroplasmas. The implication of a role for spiralin in adhesion to insect cells was further supported by adhesion assays showing that a spiralin-less mutant was impaired in adhesion and that recombinant spiralin triggered adhesion of latex beads. We also showed that cytochalasin D induced changes in the surface-exposed glycoconjugates, as inferred from the lectin binding patterns, and specifically improved adhesion of S. citri wild-type but not of the spiralin-less mutant. These results indicate that cytochalasin D exposes insect cell receptors of spiralin that are masked in untreated cells. In addition, competitive adhesion assays with lectins strongly suggest spiralin to exhibit glycoconjugate binding properties similar to that of the Vicia villosa agglutinin (VVA) lectin., (© 2014 John Wiley & Sons Ltd.)

The ultra- and microstructure of the female reproductive system of Matsucoccus matsumurae was studied using light microscopy, scanning and transmission electron microscopy. The results revealed that the female reproductive system of M. matsumurae is composed of a pair of ovaries, a common oviduct, a pair of lateral oviducts, a spermatheca and two pairs of accessory glands. Each ovary is composed of approximately 50 telotrophic ovarioles that are devoid of terminal filaments. Each ovariole is subdivided into an apical tropharium, a vitellarium and a short pedicel connected to a lateral oviduct. The tropharium contains 8-10 trophocytes and two early previtellogenic oocytes termed arrested oocytes. The trophocytes degenerate after egg maturation, and the arrested oocytes are capable of further development. The vitellarium contains 3-6 oocytes of different developmental stages: previtellogenesis, vitellogenesis and choriogenesis. The surface of the vitellarium is rough and composed of a pattern of polygonal reticular formations with a center protuberance. The oocyte possesses numerous yolk spheres and lipid droplets, and is surrounded by a mono-layered follicular epithelium that becomes binucleate at the beginning of vitellogenesis. Accessory nuclei are observed in the peripheral ooplasm during vitellogenesis., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

High temperature often induces oxidative stress and antioxidant response in insects. This phenomenon has been well documented under controlled laboratory conditions, but whether it happens under fluctuating field conditions is largely unknown. In this study, we used an invasive lace bug (Corythucha ciliata) as a model species to compare the effects of controlled thermal treatments (2 h at 33-43 °C with 2 °C intervals in the laboratory) and naturally fluctuating thermal conditions (08:00-14:00 at 2-h intervals (29.7-37.2 °C) on a hot summer day in a field in Shanghai, China) on lipid peroxidation (malondialdehyde (MDA) was the marker) and anaerobic respiration (lactate dehydrogenase (LDH) was the marker), as well as superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and glutathione reductase (GR). The results show that MDA concentration increased significantly in response to heat stresses with similar trend in the laboratory and field. LDH activities did not significantly vary across temperatures in the laboratory-exposed individuals, but they significantly increased by rising temperature in the field. The activities or concentrations of SOD, CAT, GSH, and GR all significantly increased with increasing temperature in the two populations. These findings indicate that high temperature induces oxidative stress, resulting in high anaerobic respiration and antioxidant defenses in C. ciliata under both the laboratory and field conditions, which likely provide a defense mechanism against oxidative damage due to the accumulation of ROS.

The rice brown planthopper (BPH), Nilaparvata lugens (Stål), is one of the most destructive insect pests of rice in Asia. Although resistant rice varieties can be effective in managing planthopper populations, BPH has repeatedly been able to overcome resistant rice varieties. It is possible that BPH adaptation to resistant rice varieties may be related to its endosymbionts. We investigated methods for culturing BPH cells in order to study in-depth interactions between rice, BPH, and its endosymbionts. In this study, we tested EX-CELL™ 405, EX-CELL™ 420, Mitsuhashi and Maramorosch's medium, and Kimura's medium, for in vitro culture of BPH cells. Only Kimura's medium was found to be suitable for BPH cell culture, and BPH embryos around blastokinetic stage were the best source for BPH cell culture. Cells from BPH embryonic tissues adhered to the plate substrate, and the migration of cells was observed within 24 h in primary culture. After 3 mo of subculture, various types of BPH cells were successfully maintained in the Kimura's medium.

External and internal head structures of Coleorrhyncha, a key-taxon within the Hemiptera, are described in detail and documented using modern techniques. The main focus is on Hackeriella veitchi, but two additional representatives of the Gondwanan relict group were also examined, and also head structures of Enicocephalidae, a member of a potentially basal heteropteran lineage. Features were compared to those documented in literature for the Sternorrhyncha, Auchenorrhyncha, and Heteroptera. Coleorrhyncha are characterized by highly modified head structures and correspondingly an entire series of autapomorphies, such as for instance a strongly flattened head capsule with fenestrations. However, they also display features that are likely plesiomorphic compared to members of other hemipteran groups. These include the almost complete tentorium and the lack of the gula. The sistergroup relationship between Coleorrhyncha and Heteroptera is well supported by cephalic features. Potential synapomorphies are the presence of a distinct mandibular sulcus, the reduced number of antennomeres, the absence of clasping organs in the labial groove, coiled accessory salivary ducts, the presence of a small cervical muscle M1a (M. pronotopostoccipitalis medialis), the presence of a second mandibular promotor M14 (M. zygomaticus mandibulae), the presence of M28 (M. verticopharyngalis), and M30 (M. frontobuccalis posterior)., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) cryptic species complex of whiteflies contains two species, MEAM1 and MED, that are highly invasive in supportive climates the world over. In the United States, MEAM1 occurs both in the field and in the greenhouse, but MED is only found in the greenhouse. To make inferences about the population structure of both species, and the origin and recent spread of MED within the United States, 987 MEAM1 whiteflies and 340 MED whiteflies were genotyped at six and seven microsatellite loci, respectively, for population genetic analyses. Major results of the study are 1) MED exhibits more population structure and genetic differentiation than MEAM1, 2) nuclear microsatellite markers exhibit a high degree of concordance with mitochondrial markers recovering a major east-west phylogeographic break within MED, 3) both eastern and western MED are found throughout the continental United States and eastern MED is present in Hawaii, and 4) MEAM1 contains two greenhouse U.S. populations significantly differentiated from other U.S. MEAM1. The results suggest that MED was introduced into the United States on at least three occasions and rapidly spread throughout the United States, showing no discernible differentiation across 7,000 km. The results further suggest that there is an enhanced role of the protected agricultural environment in promoting genetic differentiation in both invasive B. tabaci cryptic species.

A transmission electron-microscopy study has been carried out on the pedicel of three homopteran species, with particular focus on the leafhopper Scaphoideus titanus Ball. The two other species, the planthoppers Hyalesthes obsoletus Signoret and Metcalfa pruinosa Say, were investigated in order to compare the ultrastructure of the Johnston's organ (JO) among representatives of the Auchenorrhyncha group. The results showed the presence of a well developed JO located within the pedicel. Depending on the species the JO is made of 25 up to 72 scolopidia arranged in a coronal array. Each scolopidium is connective, heterodynal, amphinematic and hosts three structurally dissimilar sensory neurons. Two of them have a type 1 ciliary segment while the third bears a type 2 cilium. The type 2 dendrite tip is associated with a tubular cap and is longer than the others, ending into the cuticle at the base of the flagellum. Other scolopidia with one or two neurons were found in S. titanus, forming an accessory organ. The presence of such a well developed mechanosensory apparatus is discussed in relation with the lifestyle of the three species., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: The white backed planthopper (WBPH), Sogatella furcifera (Horváth), causes great damage to many crops by direct feeding or transmitting plant viruses. Southern rice black-streaked dwarf virus (SRBSDV), transmitted by WBPH, has become a great threat to rice production in East Asia., Methodology/principal Findings: By de novo transcriptome assembling and massive parallel pyrosequencing, we constructed two transcriptomes of WBPH and profiled the alternation of gene expression in response to SRBSDV infection in transcriptional level. Over 25 million reads of high-quality DNA sequences and 81388 different unigenes were generated using Illumina technology from both viruliferous and non-viruliferous WBPH. WBPH has a very similar gene ontological distribution to other two closely related rice planthoppers, Nilaparvata lugens and Laodelphax striatellus. 7291 microsatellite loci were also predicted which could be useful for further evolutionary analysis. Furthermore, comparative analysis of the two transcriptomes generated from viruliferous and non-viruliferous WBPH provided a list of candidate transcripts that potentially were elicited as a response to viral infection. Pathway analyses of a subset of these transcripts indicated that SRBSDV infection may perturb primary metabolism and the ubiquitin-proteasome pathways. In addition, 5.5% (181 out of 3315) of the genes in cell cytoskeleton organization pathway showed obvious changes. Our data also demonstrated that SRBSDV infection activated the immunity regulatory systems of WBPH, such as RNA interference, autophagy and antimicrobial peptide production., Conclusions/significance: We employed massively parallel pyrosequencing to collect ESTs from viruliferous and non-viruliferous samples of WBPH. 81388 different unigenes have been obtained. We for the first time described the direct effects of a Reoviridae family plant virus on global gene expression profiles of its insect vector using high-throughput sequencing. Our study will provide a road map for future investigations of the fascinating interactions between Reoviridae viruses and their insect vectors, and provide new strategies for crop protection.

Spiroplasma citri is a plant pathogenic mollicute transmitted by the leafhopper vector Circulifer haematoceps. Successful transmission requires the spiroplasmas to cross the intestinal epithelium and salivary gland barriers through endocytosis mediated by receptor-ligand interactions. To characterize these interactions we studied the adhesion and invasion capabilities of a S. citri mutant using the Ciha-1 leafhopper cell line. S. citri GII3 wild-type contains 7 plasmids, 5 of which (pSci1 to 5) encode 8 related adhesins (ScARPs). As compared to the wild-type strain GII3, the S. citri mutant G/6 lacking pSci1 to 5 was affected in its ability to adhere and enter into the Ciha-1 cells. Proteolysis analyses, Triton X-114 partitioning and agglutination assays showed that the N-terminal part of ScARP3d, consisting of repeated sequences, was exposed to the spiroplasma surface whereas the C-terminal part was anchored into the membrane. Latex beads cytadherence assays showed the ScARP3d repeat domain (Rep3d) to be involved, and internalization of the Rep3d-coated beads to be actin-dependent. These data suggested that ScARP3d, via its Rep3d domain, was implicated in adhesion of S. citri GII3 to insect cells. Inhibition tests using anti-Rep3d antibodies and competitive assays with recombinant Rep3d both resulted in a decrease of insect cells invasion by the spiroplasmas. Unexpectedly, treatment of Ciha-1 cells with the actin polymerisation inhibitor cytochalasin D increased adhesion and consequently entry of S. citri GII3. For the ScARPs-less mutant G/6, only adhesion was enhanced though to a lesser extent following cytochalasin D treatment. All together these results strongly suggest a role of ScARPs, and particularly ScARP3d, in adhesion and invasion of the leafhopper cells by S. citri.

Phytoplasmas, uncultivable phloem-limited phytopathogenic wall-less bacteria, represent a major threat to agriculture worldwide. They are transmitted in a persistent, propagative manner by phloem-sucking Hemipteran insects. Phytoplasma membrane proteins are in direct contact with hosts and are presumably involved in determining vector specificity. Such a role has been proposed for phytoplasma transmembrane proteins encoded by circular extrachromosomal elements, at least one of which is a plasmid. Little is known about the interactions between major phytoplasma antigenic membrane protein (Amp) and insect vector proteins. The aims of our work were to identify vector proteins interacting with Amp and to investigate their role in transmission specificity. In controlled transmission experiments, four Hemipteran species were identified as vectors of "Candidatus Phytoplasma asteris", the chrysanthemum yellows phytoplasmas (CYP) strain, and three others as non-vectors. Interactions between a labelled (recombinant) CYP Amp and insect proteins were analysed by far Western blots and affinity chromatography. Amp interacted specifically with a few proteins from vector species only. Among Amp-binding vector proteins, actin and both the α and β subunits of ATP synthase were identified by mass spectrometry and Western blots. Immunofluorescence confocal microscopy and Western blots of plasma membrane and mitochondrial fractions confirmed the localisation of ATP synthase, generally known as a mitochondrial protein, in plasma membranes of midgut and salivary gland cells in the vector Euscelidius variegatus. The vector-specific interaction between phytoplasma Amp and insect ATP synthase is demonstrated for the first time, and this work also supports the hypothesis that host actin is involved in the internalization and intracellular motility of phytoplasmas within their vectors. Phytoplasma Amp is hypothesized to play a crucial role in insect transmission specificity.

Background: Plant-pathogenic begomoviruses have a complex association with their insect vectors. The interactions of begomoviruses and reproduction of their vectors are poorly understood. Bemisia tabaci is known to transmit many begomoviruses, and the spread of B. tabaci, especially the B and Q 'biotypes', has been accompanied by the epidemics of begomoviruses. One of these identified disease-causing agents was Tomato yellow leaf curl China virus (TYLCCNV)., Methodology/principal Findings: In this study, we compared the egg production and realized fecundity of two 'biotypes' or putative species of the whitefly B. tabaci, including the alien invasive B and the indigenous ZHJ1 from Zhejiang, China, feeding on either healthy or TYLCCNV-infected tobacco plants. The ovary of the whitefly was composed of 12-22 telotrophic ovarioles. According to the morphology of the oocytes and level of yolk content, oocytes in ovarioles were divided into four developmental phases (I-IV). Significantly higher proportion of immature oocytes (phase II, III) and mature oocytes (phase IV) was observed in ovary of females that fed on TYLCCNV-infected tobacco compared to that on healthy plants. Moreover, there was significant increase of eggs laid of B whitefly that fed on TYLCCNV-infected tobacco plants during the early developmental stages. In contrast, the proportion of oocytes of different developmental phases and eggs laid had no significant differences between ZHJ1 whiteflies feeding on TYLCCNV-infected and non-infected host plants., Conclusions/significance: The invasive B whitefly benefits from feeding on a begomovirus-infected plant through increased egg production and realized fecundity.

The subcellular distributions of six digestive and non-digestive enzymes (alpha-glucosidase, beta-glucosidase, alkaline phosphatase, acid phosphatase, aminopeptidase and lactate dehydrogenase) of Eurygaster integriceps have been studied. The subcellular distributions of acid phosphatase and alpha-glucosidase are similar and the gradient ultracentrifugation profiles of these two enzymes overlap. Two partially membrane-bound enzymes, alkaline phosphatase and beta-glucosidase have similar distributions in differential centrifugation fractions, which are different from that of alpha-glucosidase. Sucrose gradient ultracentrifugation of membranes from luminal contents showed that beta-glucosidase carrying membranes are heavier. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the profile of proteins extracted from beta-glucosidase carrying membranes is different from that of alpha-glucosidase carrying membranes. We conclude that beta-glucosidase and aminopeptidase are markers of microvillar membrane (MM) and perimicrovillar space, respectively, while alpha-glucosidase and acid phosphatase are perimicrovillar markers. In E. integriceps V1 luminal content is a rich source of PMM and MM and that is used to resolve these membranes., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Using transmission electron microscopy, light microscopy (Giemsa May-Grumwald), and the Periodic Acid-Schif (PAS) and Sudan Black B staining techniques, hemocytes in the hemolymph of adult female Dactylopius coccus were characterized. The following, in order of abundance, were found: granulocytes, plasmatocytes, prohemocytes, and oenocytoids. Granulocytes varied in size with granulations in the cytoplasm, a large quantity of mitochondria, rugose endoplasmatic reticulum, ribosomes and vesicles, central or exocentric, spherical and occasionally lobulate nucleus. Plasmatocytes were polymorphic with irregularities in the plasma membrane; cytoplasm contained mitochondria, rugose endoplasmatic reticulum and vesicles, and exocentric, spherical, or irregular nucleus. In both types of hemocytes, scant polysaccharides and lipids were found. Prohemocytes were small and spherical with homogeneous cytoplasm and large exocentric nuclei. Oenocytoids were oval or irregular with dense homogeneous cytoplasm and elongated exocentric nuclei. The percentages of granulocytes on different days (d 1 and 10) during the life of the adult female were significantly different, as were those of plasmatocytes on d 30 and 50 and prohemocytes on d 1 and 50., ((c) 2010 Wiley Periodicals, Inc.)

The Asian citrus psyllid (AsCP), Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is a highly competent vector of the phloem-inhabiting bacterium Candidatus Liberibacter asiaticus associated with the citrus disease huanglongbing (HLB). Commonly referred to as citrus greening disease in the USA, HLB causes reduced fruit yields, quality, and ultimately tree death and is considered the most serious citrus disease. HLB has become a major limiting factor to the production of citrus worldwide. Studies of HLB have been impeded by the fact that C. Liberibacter has not yet been cultured on artificial nutrient media. After being acquired by a psyllid, C. Liberibacter asiaticus is reported to replicate within the psyllid and is retained by the psyllid throughout its life span. We therefore hypothesized that C. Liberibacter asiaticus could be cultured in vitro using psyllid cell cultures as the medium and investigated the establishment of a pure culture for AsCP cells. Several commercially available insect cell culture media along with some media we developed were screened for viability to culture cells from AsCP embryos. Cells from psyllid tissues adhered to the plate and migration was observed within 24 h. Cells were maintained at 20 degrees C. We successfully established primary psyllid cell cultures, referred to as DcHH-1, for D. citri Hert-Hunter-1, with a new media, Hert-Hunter-70.

Most of the meiotic literature is based on species with monocentric chromosomes, however meiosis in protoctist, plant and animal species with holocentric chromosomes is less characterized. In some cases, an inverted meiotic sequence is claimed to occur, in which segregation of homologs is postponed until the second meiotic division. Additionally, other features also deserve interest, namely: (i) the different behavior of sex chromosomes if compared to that of the autosomes; (ii) the absence of a canonical kinetochore structure; (iii) the restriction of the kinetic activity to the chromosomal ends; (iv) the variations in the orientation of bivalents at the division plate, and (v) the possible occurrence of chiasma terminalization. Here we summarize the current knowledge on these topics in the meiosis of Hemiptera (Heteroptera) and present novel results that illustrate some of the special features mentioned above. We also point out the necessity of reviewing the term 'inverted meiosis' and propose some future prospects to study this peculiar meiosis.

Rice dwarf virus (RDV) spreads, along actin-based filopodia, as one of the ways of its transport, from infected to non-infected cells of its vector insect via tubules that are composed of the viral nonstructural protein Pns10 and contain viral particles. An inhibitor that perturbs the endomembrane system, namely, brefeldin A, and butanedione monoxime, an inhibitor of myosin motor activity, abolished the formation of such tubules, preventing the direct cell-to-cell spread of RDV without any significant effects on the production of viral particles. The microtubule-depolymerizing drug NOC, which had a negligible effect on the formation and protrusion of such tubules from cells, did not inhibit the intercellular spread of RDV. Our observations suggest that the endomembrane system and myosin motors, but not microtubule, are required for the Pns10 tubule formation and are, therefore, involved in the secondary infection by RDV of adjacent cells.

Rhopalosiphum padi virus (RhPV) (family Dicistroviridae; genus Cripavirus) is an icosahedral aphid virus with a 10kb positive-sense RNA genome. To study the molecular biology of RhPV, identification of a cell line that supports replication of the virus is essential. We screened nine cell lines derived from species within the Lepidoptera, Diptera and Hemiptera for susceptibility to RhPV following RNA transfection. We observed cytopathic effects (CPE) only in cell lines derived from hemipterans, specifically GWSS-Z10 cells derived from the glassy winged sharp shooter, Homalodisca coagulata and DMII-AM cells derived from the corn leaf hopper, Dalbulus maidis. Translation and appropriate processing of viral gene products, RNA replication and packaging of virus particles in the cytoplasm of GWSS-Z10 cells were examined by Western blot analysis, Northern blot hybridization and electron microscopy. Infectivity of the GWSS-Z10 cell derived-virus particles to the bird cherry-oat aphid, R. padi, was confirmed by RT-PCR and Western blot. The GWSS-Z10 cell line provides a valuable tool to investigate replication, structure and assembly of RhPV.

The present study aimed describing the ovaries of the sugarcane spittlebug Mahanarva fimbriolata which are meroistic telotrophic with nurse cells and oocytes located in the tropharium. SEM revealed paired ovaries located dorsolaterally around the intestine, and oocytes exhibiting shapes ranging from round (less developed) to elliptic (more developed), suggesting a simultaneous, although, asynchronous development. Based on histological data we classified the oocytes in stages from I to V. Stage I oocytes exhibit follicular epithelium with cubic and/or prismatic cells, fine cytoplasmic granules. Stage II oocytes present intercellular spaces in the follicular epithelium due to the incorporation of yolk elements from the hemolymph. Small granules are present in the periphery of oocytes while larger granules are observed in the center. Stage III oocytes are larger and intercellular spaces in the follicular epithelium are evident, as well as the interface between follicular epithelium and oocyte. Yolk granules of different sizes are present in the cytoplasm. During this stage, chorion deposition initiates. Stage IV oocytes exhibit squamous follicular cells and larger intercellular spaces when compared to those observed in the previous stage. The oocyte cytoplasm present granular and viscous yolk, the latter is the result of the breakdown of granules. Stage V oocytes exhibit a follicular epithelium almost completely degenerated, smaller quantities of granular yolk and large amounts of viscous yolk. Based on our findings we established the sequence of yolk deposition in M. fimbriolata oocyte as follows: proteins and lipids, which are first produced by endogenous processes in stages I and II oocytes. Exogenous incorporation begins in stage III. In stages I and II oocytes, lipids are also produced by follicular epithelial cells. The third element to be deposited is polysaccharides, mainly found as complexes. Therefore, the yolk present in the oocytes of this species consists of glycolipoproteins. Molecular weights of proteins present in M. fimbriolata oocytes ranged from 10 to 92 KDa, differently from vitellogenin, the most common protein present in insect oocytes, weighing approximately 180 KDa.

Meiotic karyotypes in males of 16 species (assigned to 9 genera and 7 subfamilies) ofthe family Psyllidae and 3 species (assigned to 3 genera of the subfamily Triozinae) of the family Triozidae are described for the first time. The first data on the genus Ligustrinia are presented. All the species were shown to exhibit the modal karyotype for psyllids, 2n = 24 + X, except Bactericera nigricornis and Arytainilla spartiophila, in which 2n = 24 + XY and 2n = 22 + X were found, respectively. The karyotype of Ctenarytaina eucalypti (Psyllidae, Spondyliaspidinae) was reinvestigated, and the karyotype 2n = 10 + X, characteristic of Spondyliaspidinae, was revealed. The karyotypes ofStrophingia fallax, S. arborea, and Craspedolepta topicalis were studied using the C-banding technique.

Mature spermatozoa from five species of cicadas of the subfamily Cicadettinae (Quintilia wealei, Melampsalta leucoptera, Stagira simplex, Xosopsaltria thunbergi and Monomatapa matoposa) were examined by light and electron microscopy. In each species sperm are elongate, aggregated into organized bundles with their heads embedded in a homogenous matrix to form spermatodesmata, and exhibit polymegaly. The head of the sperm consist of an anteriorly positioned conical acrosome that has a tubular substructure and a deep, posterior invagination that forms the subacrosomal space (eccentrically positioned anteriorly). The acrosome is flattened anteriorly; posteriorly it extends along either side of the nucleus as two tubular processes that gradually decrease in diameter. The filiform nucleus tapers anteriorly and intrudes into the subscrosomal space. Posteriorly the nucleus has a lateral invagination that houses material of the so-called centriolar adjunct. Posterior to the centriolar adjuct and the nucleus are two crystalline mitochondrial derivatives and a centriole, respectively, the latter giving rise to the axoneme, which has a 9 + 9 + 2 arrangement of microtubules. In these respects the sperm are similar to those of platypleurine cicadas. However, some features seem unique to cicadettines, including the structural organization of an enlarged centriolar adjunct and the dimensions of the tails. The enlarged centriolar adjunct has a lamella-like substructure and can be considered a synapomorphic character in the Cicadettinae. It is, therefore, potentially useful in the separation of this subfamily from the Cicadinae. In addition, the great length of the sperm nucleus of long-headed sperm in M. matoposa could be a synapomorphy of this genus and related taphurine and cicadettine species.

The insect body plan is very well conserved, yet the developmental mechanisms of segmentation are surprisingly varied. Less evolutionarily derived insects undergo short germ segmentation where only the anterior segments are specified before gastrulation whereas the remaining posterior segments are formed during a later secondary growth phase. In contrast, derived long germ insects such as Drosophila specify their entire bodies essentially simultaneously. These fundamental embryological differences imply potentially divergent molecular patterning events. Numerous studies have focused on comparing the expression and function of the homologs of Drosophila segmentation genes between Drosophila and different short and long germ insects. Here we review these comparative data with special emphasis on understanding how short germ insects generate segments and how this ancestral mechanism may have been modified in derived long germ insects such as Drosophila. We break down the larger issue of short versus long germ segmentation into its component developmental problems and structure our discussion in order to highlight the unanswered questions in the evolution of insect segmentation.

The genome of the Tenuivirus, Rice stripe virus (RSV) comprises four RNAs, the smallest three of which each contain two open reading frames (ORFs) arranged in an ambisense manner. The expression of the ORFs from RNAs 2-4 in plants and the insect vector, Laodelphax striatellus, was studied using antisera raised against the gene products. In Western blotting of the proteins from infected plants, the molecular masses of p2, p3, pc3 (nucleocapsid protein, N) and p4 (major non-structural protein, NCP) were as expected; that of pc4 appeared larger than expected. Antisera to the N- and C-terminal parts of the complementary ORF on RNA 2, analogous to that encoding glycoproteins on genomes of bunyaviruses and tospoviruses, revealed banding patterns suggestive of processing of the product; the possible processing is discussed. Four types of inclusion bodies were identified by immunofluorescent and immunogold microscopy of thin sections of infected leaves. Most electron-dense amorphous semi-electron-opaque inclusion bodies (dASO) contained only p4 while some contained at least p2, pc2-N, p3, pc3 as well as p4. A ring-like structure containing at least pc2-N, p4 and pc4 was also identified in infected plant cells. Fibrillar amorphous semi-electron-opaque inclusion bodies (fASO) contained only p4. Filamentous electron-opaque inclusion bodies (FEO), which consist of pc2-N(.)and p4, were found both in infected plant cells and in the mid-gut lumen and mid-gut epithelial cells of L. striatellus. This suggests an interaction between p4 and pc2-N and a function of pc2-N distinct from that of its-homologue in Bunyaviridae. Our results confirm the in vivo ambisense coding strategy of Tenuivirus RNA 2 and provide further evidence that RSV does not produce enveloped virions in infected rice plants.

Four continuous cell lines were established from the embryos of the glassy-winged sharpshooter, Homalodisca coagulata (Say), an economically important insect vector of bacterial pathogens of grape, almond, citrus, oleander, and other agricultural and ornamental plantings. The cell lines were designated GWSS-Z10, GWSS-Z15, GWSS-G3, and GWSS-LH. The GWSS-Z10, GWSS-Z15, and GWSS-G3 lines were cultured in Ex-Cell 401 medium supplemented with 10% fetal bovine serum (FBS), whereas the GWSS-LH line was cultured in LH medium supplemented with 20% FBS. The cell lines were characterized in terms of their morphology, growth, protein composition, and polymerase chain reaction- amplification patterns of their chromosomal deoxyribonucleic acid. The population doubling times of GWSS-Z10, GWSS-Z15, GWSS-G3, and GWSS-LH were 46.2, 90.9, 100.3, and 60.2 h, respectively. These lines should be useful for the study of insect-pathogenic viruses of leafhoppers, aphids, treehoppers, and other related insects as well as plant-pathogenic viruses that are transmitted by these insects.

Selected morphological and physiological properties of the corpus allatum (CA)-corpus cardiacum (CC) complex from the two-spotted stinkbug, Perillus bioculatus (Hemiptera: Pentatomidae), were studied. The CAs play an important role in insect physiology because of their production of the juvenile hormones (JHs), i.e., key hormones involved in development and reproduction. We found that the P. bioculatus CA-CC complex is present in two distinct morphological forms, the more frequently observed complex containing one "fused" CA between two CCs and the more rarely observed complex having one CA laterally attached to each CC. These complexes were tested for their ability to synthesize JH-like compounds. We found that the primary lipophilic compound synthesized by the CA-CCs migrated differently from JH III (a JH found in numerous insect species) when subjected to thin-layer chromatography. Furthermore, the synthesis of this compound is stimulated by 2E,6E-farnesol, a known precursor for JH III. These data indicate that the P. bioculatus CA- CC product has chemical properties similar to that of other (as of yet unidentified) hemipteran JHs. In addition, we found that the synthesis of this product is sensitive to pH and buffer type; minimally or not affected by the absence of the CC; expressed at similar levels in days 5-30 postemergent adults; and inhibited or decreased in adults reared under low temperature-short day conditions.

The surface topography and ultrastructure of the labial cuticle of Cenocorixa bifida were examined by scanning and transmission electron microscopy. The dorsal wall of the labium consists of seven sclerotized transverse bars each displaying two rows of semicircular grooves and pores. The cuticle is about 20 microm thick and is composed of epicuticle and lamellate exocuticle and endocuticle, the latter separated from the underlying epidermis by subcuticle containing amorphous material. The epicuticle is subdivided into an electron-dense very thin outer epicuticle and a homogenous thick inner epicuticle, which is penetrated by grooves. The exocuticle is filled with electron-dense blocks of material, which may provide mechanical support to the labial wall. The elongate epidermal cells display extensive infoldings of the apical plasma membrane (facing the cuticle) and contain abundant mitochondria in the cytoplasm. The presence of deep epicuticular grooves and pores in the thin labial cuticle and extensive apical membrane infolding and abundant mitochondria in the epidermal cells suggest that the labium in C. bifida is the site of osmoregulatory ionic uptake.

Meiotic karyotypes were studied in males of Craspedolepta sonchi (Foerster, 1848), Diaphorina chobauti Puton, 1898, D. lamproptera Burckhardt, 1981, Psylla hartigii Flor, 1861, Cacopsylla palmeni (Loew, 1878), C. hippophaes (Foerster, 1848), C. melanoneura (Foerster, 1868), C. pyricola (Foerster 1848), C. moscovita (Andrianova, 1848), Bactericera salicivora (Reuter, 1876), Trioza abdominalis Flor, 1861, T. lauri = Lauritrioza alacris (Flor, 1861). Karyotypes were 2n = 25 (24 + XO) in all species except B. salicivora with 2n = 26 (24 + neo-XY). Testes consisted of two follicles each in all species but P. hartigii with four-follicular testes in males. The discussion covers the problems of chromosome numbers, sex-determining chromosome systems, B-chromosomes, patterns of C-banding, testis structure, and spermatid development in Psylloidea.

The body of the whitefly Aleurochiton aceris contains specialized cells, termed mycetocytes, that enclose endosymbiotic microorganisms. The endosymbionts are transmitted from one generation to the next transovarially. In contrast to other insects, in whiteflies whole intact mycetocytes migrate into the ovaries, traverse the follicular epithelium, and reach the oocyte surface (i.e., perivitellin space). The migration of mycetocytes begins in the last instar, called puparium, from which imagines emerge. During this stage the cytoplasm of mycetocytes is tightly packed with pleomorphic bacteria and less numerous coccoid microorganisms. In adult females the mycetocytes gather extracellularly in the depression of the vitellarial oocyte. Till the end of oogenesis neither pleomorphic nor coccoid microorganisms are released from mycetocytes into the oocyte.

To identify the means by which phytohemagglutinin (PHA) exerts its toxicity on the potato leafhopper, four different methods (thick and semi-thin sectioning combined with immunofluorescent staining, in vitro receptor autoradiography, and immunoelectron microscopy) were used to elucidate the PHA target tissue, binding site, and its effects on this tissue. Sixteen 1- or 2-day-old female potato leafhoppers were fed for 36 h on each of three treatments: a control, diet or a diet containing either the PHA-E subunit or the PHA-L subunit. The PHA-E subunit, but not PHA-L, had previously been shown to be lethal. The insects were then prepared for both light and confocal microscopy. Analysis of images showed that PHA bound only to the surface of midgut epithelial cells of the potato leafhopper. PHA-E caused severe disruption, disorganization, and elongation of the brush border microvilli, and swelling of the epithelial cells into the lumen of the gut, leading to complete closure of the lumen. Furthermore, PHA-E stimulated the division of midgut epithelial cell nuclei, leading to two nuclei in each cell. Nuclei later elongated and degraded. In contrast, PHA-L had little effect on the epithelial cells of the midgut. It did not strongly bind to the surface of epithelial cells and caused much less disruption of brush-border microvilli, less disorganization of the cells and less elongation of nuclei. Strong binding of PHA occurred solely on the cell membrane of the brush border microvilli of epithelial cells. In contrast, the controls (i.e., midgut tissue, blocking agent, PHA, and antibodies) showed that midgut tissue was not autofluorescent and showed no fluorescent binding signal. Analysis of both bright- and dark-field images obtained by autoradiography and immunoelectron microscopy confirmed these findings.

Mitosis in the hemipteran Agallia constricta (leafhopper) cell line AC-20 was examined by light microscopy of living and fixed cells. During early prometaphase the numerous small (0.30-3.0-microns) chromosomes appear as discrete units that lack a primary constriction. However, by late prometaphase the chromosomes are tightly packed at the spindle equator and are no longer clearly resolvable as individuals. When viewed from the side the metaphase chromatin appears as a 2-3-microns wide band that spans the width of the spindle; when viewed from the pole it appears as a fenestrated disk. The metaphase chromatin splits at anaphase into two sister chromatin plates, each of which exhibits holokinetic poleward movement, i.e., all parts of each plate move as a single unit with the same velocity. In many early-to-mild anaphase cells the separating sister plates are connected by chromatin-containing bridges that break as anaphase progresses. Ultrastructural analyses of serial thick and thin sections from cells fixed by conventional, OsO4/KFeCN, or high pressure rapid freezing methods, reveal that by metaphase all of the chromosomes are interconnected to form a large, irregularly shaped fenestrated disk of chromatin. Similar analyses reveal that adjacent chromatids remain interconnected throughout anaphase. Each disk of metaphase and anaphase chromatin contains numerous kinetochores recessed within its pole-facing surface. Kinetochores consist of a fine, faintly staining fibrillar material arranged along the chromatin surface as thin (0.1-0.3 micron dia.) rods varying considerably (0.15-2.3 microns) in length. From these observations we conclude that the polycentric metaphase chromatin of A. constricta, and its holokinetic behavior during anaphase, arises from the aggregation or cohesion of smaller prometaphase chromosomes, each of which contains a single, diffuse kinetochore.