Characterization of cell lines developed from the glassy-winged sharpshooter, Homalodisca coagulata (hemiptera: Cicadellidae)

Summary 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 reactionamplification 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.     

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.     

First record of Clonostachys rosea (Ascomycota: Hypocreales) as an entomopathogenic fungus of Oncometopia tucumana and Sonesimia grossa (Hemiptera: Cicadellidae) in Argentina

Clonostachys rosea (Link: Fries) Schroers, Samuels, Seifert, and Gams (Ascomycota: Hypocreales) has been reported as a mycoparasite of fungi and nematodes and as saprobe in soils, but this fungus has not been reported previously to be entomopathogenic. Many species of cicadellid leafhoppers cause economic damage to crops as vectors of plant pathogens. In the present work, we report the first record of C. rosea as an entomopathogenic fungus of two leafhoppers pest, Oncometopia tucumana and Sonesimia grossa (Hemiptera: Cicadellidae), in Argentina and evaluate the pathogenicity of C. rosea against them.     

Stylet penetration of Cacopsylla pyri; an electrical penetration graph (EPG) study

Detailed information on plant penetration activities by pear psylla Cacopsylla pyri L. (Hemiptera Psyllidae) is essential to study phytoplasma transmission of “Candidatus Phytoplasma pyri” responsible of pear decline disease (PD) and to trace and evaluate resistant traits in new pear tree selections for advanced breeding programs. The electrical penetration graph technique or (full) EPG may relevantly contribute to this knowledge. C. pyri EPG waveforms were characterized on basis of amplitude, frequency, voltage level, and electrical origin. Additionally, stylet tracks and the putative location of stylet tips in the plant tissue were histologically related to EPG waveforms by light and transmission electron microscopy observations after stylectomy. More than one waveform occurred in the same tissue: PA, PB, PC1 and PC2 were all detected in the mesophyll, and PE1 and PE2 were both recorded in the phloem. Waveform PE1 was always preceded by transient waveform PD, as previously described in other psyllids. Interestingly, no brief intracellular punctures (potential drop waveforms) were observed during plant penetration, opposite of what is usually recorded in aphids and other Sternorrhyncha.     

Virulence of Hypocreales fungi to pecan aphids (Hemiptera: Aphididae) in the laboratory

There is need for efficacious biocontrol agents for aphids in commercial orchards. As a preliminary step to this end we determined the virulence of several Hypocreales fungi to pecan aphids. In the first experiment we tested the virulence of Isaria fumosorosea (ARSEF 3581) blastospores to three pecan aphids Monellia caryella, Melanocallis caryaefoliae, and Monelliopsis pecanis under laboratory conditions. Rates of 1 × 10⁷ or 1 × 10⁸ spores per ml were applied in 2 ml via a spray tower to 90 mm Petri dishes containing 10 aphids each. Mortality and mycosis were determined after 24, 48 and 72 h. Treatment effects were observed by 48 h post-application, and by 72 h the higher application rate caused >90% mortality and mycosis in M. caryella and M. caryaefoliae, whereas <70% was observed in M. pecanis.We conducted two subsequent experiments (Experiments 2 and 3), using the same methodology, to compare the virulence of several Hypocreales species and strains against the aphid of primary economic concern to most pecan growers, M. caryaefoliae. In Experiment 2, we compared blastospores and conidia of two I. fumosorosea strains (ARSEF 3581 and ATCC 20874 [= strain 97]). The blastospores of ARSEF 3581 and conidia of ATCC 20874 showed higher virulence than other treatments and thus were included in Experiment 3, which also compared the virulence of conidia of Beauveria bassiana (GHA strain) and Metarhizium anisopliae (F52 strain). Results in Experiment 3 indicated the highest virulence in I. fumosorosea 3581 blastospores and M. anisopliae (F52) followed by I. fumosorosea (20874) conidia. The detection of pathogenicity to pecan aphids establishes the potential for commercial usage and additional study. Results reported here will narrow treatments to test in future greenhouse and field trials.     

Effect of intestinal erythrocyte agglutination on the feeding performance of Triatoma brasiliensis (Hemiptera: Reduviidae)

Triatoma brasiliensis is an important vector of Trypanosoma cruzi in Brazil. The feeding efficiency on its hosts depends on several parameters including the maintenance of the ingested blood at low viscosity, which could be modulated by the anterior midgut (crop) anticoagulant and haemagglutinant activities. In the present study, we characterized T. brasiliensis crop haemagglutination activity and evaluated its importance in the feeding process. Soluble crop contents (SCC) of T. brasiliensis were able to agglutinate rat, mouse and rabbit eryhtrocytes, but had no activity on cattle and Thrichomys apereoides, a rodent species commonly associated with T. brasiliensis in the wild. The haemagglutination was characterized by the immediate formation of several clusters of erythrocytes connected by flexible elastic-like fibers. The feeding efficiency of T. brasiliensis on rat (agglutinated by SCC) was almost double that from T. apereoides (not agglutinated by SCC). The influence of haemagglutination on feeding was confirmed by artificially feeding bugs on a diet composed of cattle or rat erythrocytes. The bugs fed on cattle erythrocytes had lower ingestion rates in comparison to those fed on rats. The results indicate that, in addition to other parameters, haemagglutination brought about by SCC has an important role in the feeding efficiency of T. brasiliensis.     

Phylogenetic incongruence inferred with two mitochondrial genes in Mepraia spp. and Triatoma eratyrusiformis(Hemiptera,Reduviidae)

Mitochondrial DNA (mtDNA) is widely used to clarify phylogenetic relationships among and within species, and to determine population structure. Due to the linked nature of mtDNA genes it is expected that different genes will show similar results. Phylogenetic incongruence using mtDNA genes may result from processes such as heteroplasmy, nuclear integration of mitochondrial genes, polymerase errors, contamination, and recombination. In this study we used sequences from two mitochondrial genes (cytochrome b and cytochrome oxidase subunit I) from the wild vectors of Chagas disease, Triatoma eratyrusiformis and Mepraia species to test for topological congruence. The results showed some cases of phylogenetic incongruence due to misplacement of four haplotypes of four individuals. We discuss the possible causes of such incongruence and suggest that the explanation is an intra-individual variation likely due to heteroplasmy. This phenomenon is an independent evidence of common ancestry between these taxa.     

Morphological description of the mouthparts of the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae)

Scanning (SEM) and transmission (TEM) electron microscopy were used to elucidate the morphology of the rostrum, as well as the mandibular and maxillary stylets of the psyllid Diaphorina citri, vector of phloem-inhabiting bacteria associated with citrus huanglongbing (HLB) disease. D. citri has a cone-shaped rostrum that extends behind the pair of prothoracic coxae. The stylet bundle comprises a pair of mandibular (Md) and maxillary (Mx) stylets with a mean length of 513.3 μm; when retracted, their proximal portions form a loop and are stored in the crumena (Cr). Serial cross-sections of the rostrum revealed that the mandibles are always projected in front of the maxillary stylets. The two maxillary stylets form the food and salivary canals, with diameters of 0.9 μm and 0.4 μm respectively. These two canals merge at the end of the stylets forming a common duct with a length of 4.3 μm and a mean diameter of 0.9 μm. The acrostyle, a distinct anatomical structure present in the common duct of aphid maxillary stylets, was not observed by TEM in the ultrathin cross-sections of the common duct (CD) of D. citri. This study provides new information on D. citri mouthparts that may help to understand the feeding behaviour of this important vector of HLB-associated bacteria.     

Overexpression, purification, biochemical characterization, and molecular modeling of recombinant GDP-mannosyltransferase (GumH) from Xylella fastidiosa

The GumH enzyme from Xylella fastidiosa catalyzes the transfer reaction of a mannose from GDP-mannose to the carrier lipid cellobiose-pyrophosphate-polyprenol (Glc(2)-PP-Lip), an intermediary in the reaction for the synthesis of the exopolysaccharide (EPS) fastidian gum. The gumH gene was subcloned in the pMal-c2x vector, allowing the expression of the GumH-MBP fusion protein. Various attempts were made to obtain protein with the necessary degree of purity for crystallographic studies but the yield was very low. The gumH gene was then subcloned in the pET28a vector allowing the expression of the GumH enzyme in fusion with a histidine-rich peptide. The protein was purified and characterized. The three-dimensional structure of the X. fastidiosa GumH enzyme was modeled by threading studies. The model consists of N- and C-terminal domains similar in size and topology and separated by a deep cleft, which includes the EX(7)E motif that can be involved in the catalysis of GumH.     

Brasiliensin: A novel intestinal thrombin inhibitor from Triatoma brasiliensis (Hemiptera: Reduviidae) with an important role in blood intake

Every hematophagous invertebrate studied to date produces at least one inhibitor of coagulation. Among these, thrombin inhibitors have most frequently been isolated. In order to study the thrombin inhibitor from Triatoma brasiliensis and its biological significance for the bug, we sequenced the corresponding gene and evaluated its biological function. The T. brasiliensis intestinal thrombin inhibitor, termed brasiliensin, was sequenced and primers were designed to synthesize double strand RNA (dsRNA). Gene knockdown (RNAi) was induced by two injections of 15mug of dsRNA into fourth instar nymphs. Forty-eight hours after the second injection, bugs from each group were allowed to feed on hamsters. PCR results showed that injections of dsRNA reduced brasiliensin expression in the anterior midgut by approximately 71% in knockdown nymphs when compared with controls. The reduction in gene expression was confirmed by the thrombin inhibitory activity assay and the citrated plasma coagulation time assay which showed activity reductions of approximately 18- and approximately 3.5-fold, respectively. Knockdown nymphs ingested approximately 39% less blood than controls. In order to confirm the importance of brasiliensin in blood ingestion, fourth instar nymphs were allowed to ingest feeding solution alone or feeding solution containing 15U of thrombin prior to blood feeding. Fifty-five percent less blood was ingested by nymphs which were fed thrombin prior to blood feeding. The results suggest that anticoagulant activity in the midgut is an important determinant of the amount of blood taken from the host. The role of anticoagulants during blood ingestion is discussed in the light of this novel insight.     

Taxonomic review of the subgenus Uroleucon (Uromelan) (Hemiptera: Aphididae) in the Korean peninsula

Until now, five species of the subgenus Uroleucon (Uromelan) have been recognized in Korea. This is the first report of Uroleucon (Uromelan) adenophorae (Matsumura, 1918) occurring on Adenophora triphylla (Campanulaceae) in Gangwon-do, South Korea. Host plants are reviewed and an identification key to species is presented for six Uroleucon (Uromelan) species from the Korean Peninsula.     

Surface antigens of Xenorhabdus nematophila (F. Enterobacteriaceae) and Bacillus subtilis (F. Bacillaceae) react with antibacterial factors of Malacosoma disstria (C. Insecta: O. Lepidoptera) hemolymph

Previous research established different interactions of the insect pathogen, Xenorhabdus nematophila and nonpathogen, Bacillus subtilis, with antimicrobial hemocytes and humoral factors of larval Malacosoma disstria [Giannoulis, P., Brooks, C.L., Dunphy, G.B., Mandato, C.A., Niven, D.F., Zakarian, R.J., 2007. Interaction of the bacteria Xenorhabdus nematophila (Enterobacteriaceae) and Bacillus subtilis (Bacillaceae) with the hemocytes of larval Malacosoma disstria (Insecta: Lepidoptera: Lasicocampidae). J. Invertebr. Pathol. 94, 20-30]. The antimicrobial systems were inhibited by X. nematophila and stimulated by B. subtilis. The bacterial surface antigens participating in these reactions were unknown. Thus, herein the effects of lipopolysaccharide (endotoxin) from X. nematophila and lipoteichoic acid from B. subtilis on the larval M. disstria immune factors, the hemocytes and phenoloxidase, were determined. Endotoxin elevated the level of damaged hemocytes limiting the removal of X. nematophila from the hemolymph and enhancing the rapid release of bacteria trapped by nodulation. Similar effects were observed with the lipid A moiety of the endotoxin. The effects of lipopolysaccharide and lipid A on the hemocyte activities were abrogated by polymyxin B (an antibiotic that binds to lipid A) confirming lipopolysaccharide as the hemocytotoxin by virtue of the lipid A moiety. Lipoteichoic acid elicited nodulation and enhanced phenoloxidase activation and/or activity. Although lipoidal endotoxin and lipid A inhibited phenoloxidase activation they enhanced the activity of the enzyme. Apolipophorin-III precluded the effects of lipopolysaccharide, lipid A, and lipoteichoic acid on the hemocytes and prophenoloxidase until the antigens exceeded a critical threshold.     

Risk of Trypanosoma cruzi I (Kinetoplastida: Trypanosomatidae) transmission by Panstrongylus geniculatus (Hemiptera: Reduviidae) in Caracas (Metropolitan District) and neighboring States, Venezuela

The collection of Panstrongylus geniculatus bugs by inhabitants of dwellings in Caracas city (Metropolitan District) and in the neighboring Miranda and Vargas Sates, Venezuela, allowed for the gathering of data on the potential role of this sylvatic triatomine bug as a vector of Chagas disease in this area. The natural infection by Trypanosoma cruzi was recorded by examining fresh and stained faeces of the bugs. Additionally, a random amplification of polymorphic DNA technique for parasite identification and group typing was employed. A dot-ELISA test was used to identify the gut content of the triatomine bugs with the aim of assessing and quantifying the vector-human contact. Sixty-seven specimens (76.1%) were positive to T. cruzi (identified as T. cruzi I) and 60.2% (53/88) gave a positive reaction to the human antiserum. The human blood-positive samples included mixed blood meals with domestic animals (dog, pig and cow) (9.4%) and with mouse (3.8%). The overall Human Blood Index, measured as the percentage of bugs whose gut contents reacted with human antiserum on the total numbers of bugs that reacted with all the antisera tested, was 98.1%. Almost 41% of the bugs that had fed on humans were also positive for T. cruzi. These data show that the feeding of P. geniculatus on humans does not seem to be accidental and that its rate of infection by T. cruzi is high in this area which is not regarded as endemic for Chagas disease by the National Control Programme. This situation is particularly striking because it occurs in and around Caracas, the capital city, where 20% of the whole population of Venezuela live, human migrations from endemic areas are continuous, people in the crowded shantytown as well as people living in high-quality country houses are equally at risk and the epidemiological cycle Didelphis marsupialis/Rattus rattus-P. geniculatus-human does appear to occur successfully.     

Genetics and taxonomy of Chilean smooth-shelled mussels, Mytilus spp. (Bivalvia: Mytilidae)

It has been previously established that native smooth-shelled mussels in southern South America possess close evolutionary affinities with Northern-Hemisphere Mytilus edulis L. 1758 (McDonald et al. (1991) [5]). This result has since been challenged by authors claiming that Chilean mussels should be considered a local subspecies of M. galloprovincialis Lmk. 1819. Moreover, morphological, physiological, ecotoxicological and molecular genetic studies on Chilean smooth-shelled mussels still frequently refer to ‘M. chilensis’ Hupé 1854, even though the previous discovery of alien M. galloprovincialis and considerable heterogeneity in shell morphology among samples collected along the Chilean shores raise concerns that different Mytilus spp. species might have been included under ‘M. chilensis’. Here we reviewed the molecular and morphological data available on smooth-shelled mussels from Chile in an attempt to clarify both their genetic composition and their taxonomic status. Using multivariate analysis on sample × allozyme-frequency matrices, we confirmed the widespread occurrence of the Southern-Hemisphere form of M. edulis along the shores from the North Patagonia region of Chile to the southern tip of the South American continent. The populations sampled in southern central Chile showed some evidence of slight introgression from Southern-Hemisphere M. galloprovincialis. Morphological characterization of a sample from Dichato in southern central Chile was consistent with its previous genetic identification as Mediterranean M. galloprovincialis. The occurrence of Southern-Hemisphere M. galloprovincialis in Punta Arenas at the southern tip of the South American continent was also reported. Southern-Hemisphere M. edulis, including native Chilean smooth-shelled Mytilus, should be assigned subspecific rank and named M. edulis platensis d’Orbigny 1846.     

Identification of Mediterranean Diplodus spp. and Dentex dentex (Sparidae) by means of DNA Inter-Simple Sequence Repeat (ISSR) markers

The aim of this paper was to test the effectiveness of Inter-Simple Sequence Repeat (ISSR) technique in detecting specific molecular markers to genetically discriminate some species of teleost fish. As model species, we used four Mediterranean sea bream species (i.e. Diplodus annularis, D. puntazzo, D. sargus sargus, and D. vulgaris), and the common Mediterranean dentex Dentex dentex. Furthermore, the occurrence in our samples of two partially albino sparids (suspected, on a morphological basis, to probably belong to D. sargus sargus and to D. dentex, respectively) allowed ISSRs to be tested on doubtful specimens. Eight ISSR primers produced 95 out of 97 scorable polymorphic loci with a large number (63) of species-private bands. Maximum parsimony analysis, ΔK statistics, and assignment test were consistent in separating the five species and solving the taxonomic position of specimens of dubious attribution. The results obtained evidence the usefulness of ISSR markers in specimen and species identification, particularly when the morphological traits of fish make the classification problematic.     

Fate of resting cysts of Alexandrium spp. ingested by Perinereis nuntia (Polychaeta) and Theola fragilis (Mollusca)

The ingestion of resting cysts of Alexandrium spp. by Perinereis nuntia (Polychaeta) and Theola fragilis (Mollusca) was experimentally examined in the laboratory. P. nuntia and T. fragilis were cultured in bottom sediment containing a high density of Alexandrium cysts under dark conditions. Moreover, to evaluate the degree and consequence of being ingested, the density of cysts in the control sediment (no macrobenthic organisms) and the germination capability of the cysts in the faecal pellets of the two species of macrobenthos were examined.Cysts in the culture sediment were found to be ingested by both P. nuntia and T. fragilis. No difference in the density of cysts between the sediments cultured with and without P. nuntia was observed. However, the density of cysts in the sediments with T. fragilis decreased by 24% compared to the density in the control sediment. It is possible that most of the cysts ingested were digested by T. fragilis. The rate of Alexandrium cyst digestion by this species is estimated 594 cysts/individual/day. It is estimated that 91% of the cysts ingested by T. fragilis were partially or totally digested and only 9% were excreted in a viable state during the experiment. Thus, T. fragilis has a stronger affect on the abundance of Alexandrium cysts compared with P. nuntia.No significant difference was observed between the germination success of the cysts from faecal pellets of P. nuntia and T. fragilis compared to the cysts in the control sediment. If, however, the necessary light for the cysts to germinate is cut off by being enclosed within the faecal pellet, the germination rate of cysts from the faecal pellets may be suppressed.     

New bioassay method to assess the pathogenicity of Colombian strains of Metarhizium anisopliae (Metsch.) Sorokin and Paecilomyces sp. (Deuteromycotina: Hyphomycetes) against the subterranean burrower bug Cyrtomenus bergi Froeschner (Hemiptera: Cydnidae)

Using a new sand bioassay method, the pathogenicity of three Colombian strains of Metarhizium anisopliae (i.e., CIAT 224, 245, and 230) and one strain of Paecilomyces sp. (CIAT 308) were tested against fourth instars of the subterranean burrower bug Cyrtomenus bergi Froeschner (Hem.: Cydnidae) in the laboratory. The proposed bioassay simulates the habitat of C. bergi and at the same time enables the evaluation of a large number of insects in a short period of time and under controlled conditions. All tested fungal species/strains were pathogenic against C. bergi. However, only low levels of mortality were obtained, and never exceeded 55%. In the screening experiment, M. anisopliae strains CIAT 224 and CIAT 245 caused the highest levels of mortality. The use of the bioassay procedure described herein is discussed.     

Characterization of Beauveria bassiana and Metarhizium anisopliae isolates for management of tarnished plant bug,Lygus lineolaris (Hemiptera: Miridae)

Selected morphological and physiological characteristics of four Beauveria bassiana (Balsamo) Vuillemin isolates and one Metarhizium anisopliae (Metschnikoff) Sorokin isolate, which are highly pathogenic to Lygus lineolaris (Palisot de Beauvois) (Hemiptera: Miridae), were determined. There were significant differences in conidial size, viability, spore production, speed of germination, relative hyphal growth, and temperature sensitivity. Spore viability after incubation for 24h at 20 degrees C ranged from 91.4 to 98.6% for the five isolates tested. Spore production on quarter-strength Sabouraud dextrose agar plus 0.25% (w/v) yeast extract after 10 days incubation at 20 degrees C ranged from 1.6x10(6) to 15.5x10(6)conidia/cm(2). One B. bassiana isolate (ARSEF 1394) produced significantly more conidia than the others. Spore germination was temperature-dependant for both B. bassiana and M. anisopliae. The time required for 50% germination (TG(50)) ranged from 25.0 to 30.9, 14.0 to 16.6, and 14.8 to 18.0h at 15, 22, and 28 degrees C, respectively. Only the M. anisopliae isolate (ARSEF 3540) had significant spore germination at 35 degrees C with a TG(50) of 11.8h. A destructive sampling method was used to measure the relative hyphal growth rate among isolates. Exposure to high temperature (40-50 degrees C) for 10min had a negative effect on conidial viability. The importance of these characteristics in selecting fungal isolates for management of L. lineolaris is discussed.