If host is refractory, insistent parasite goes berserk: Trypanosomatid Blastocrithidia raabei in the dock bug Coreus marginatus.

Here we characterized the development of the trypanosomatid Blastocrithidia raabei in the dock bug Coreus marginatus using light and electron microscopy. This parasite has been previously reported to occur in the host hemolymph, which is rather typical for dixenous trypanosomatids transmitted to a plant or vertebrate with insect's saliva. In addition, C. marginatus has an unusual organization of the intestine, which makes it refractory to microbial infections: two impassable segments isolate the anterior midgut portion responsible for digestion and absorption from the posterior one containing symbiotic bacteria. Our results refuted the possibility of hemolymph infection, but revealed that the refractory nature of the host provokes very aggressive behavior of the parasite and makes its life cycle more complex, reminiscent of that in some dixenous trypanosomatids. In the pre-barrier midgut portion, the epimastigotes of B. raabei attach to the epithelium and multiply similarly to regular insect trypanosomatids. However, when facing the impassable constricted region, the parasites rampage and either fiercely break through the isolating segments or attack the intestinal epithelium in front of the barrier. The cells of the latter group pass to the basal lamina and accumulate there, causing degradation of the epitheliocytes and thus helping the epimastigotes of the former group to advance posteriorly. In the symbiont-containing post-barrier midgut segment, the parasites either attach to bacterial cells and produce cyst-like amastigotes (CLAs) or infect enterocytes. In the rectum, all epimastigotes attach either to the cuticular lining or to each other and form CLAs. We argue that in addition to the specialized life cycle B. raabei possesses functional cell enhancements important either for the successful passage through the intestinal barriers (enlarged rostrum and well-developed Golgi complex) or as food reserves (vacuoles in the posterior end).

Insecticidal activity of the metalloprotease AprA occurs through suppression of host cellular and humoral immunity.

The biochemical characterization of virulence factors from entomopathogenic bacteria is important to understand entomopathogen-insect molecular interactions. Pseudomonas entomophila is a typical entomopathogenic bacterium that harbors virulence factors against several insects. However, the molecular actions of these factors against host innate immune responses are not clearly elucidated. In this study, we observed that bean bugs (Riptortus pedestris) that were injected with P. entomophila were highly susceptible to this bacterium. To determine how P. entomophila counteracts the host innate immunity to survive within the insect, we purified a highly enriched protein with potential host insect-killing activity from the culture supernatant of P. entomophila. Then, a 45-kDa protein was purified to homogeneity and identified as AprA which is an alkaline zinc metalloprotease of the genus Pseudomonas by liquid chromatography mass spectrometry (LC-MS). Purified AprA showed a pronounced killing effect against host insects and suppressed both host cellular and humoral innate immunity. Furthermore, to show that AprA is an important insecticidal protein of P. entomophila, we used an aprA-deficient P. entomophila mutant strain (ΔaprA). When ΔaprA mutant cells were injected to host insects, this mutant exhibited extremely attenuated virulence. In addition, the cytotoxicity against host hemocytes and the antimicrobial peptide-degrading ability of the ΔaprA mutant were greatly decreased. These findings suggest that AprA functions as an important insecticidal protein of P. entomophila via suppression of host cellular and humoral innate immune responses.

A midgut lysate of the Riptortus pedestris has antibacterial activity against LPS O-antigen-deficient Burkholderia mutants.

Riptortus pedestris, a common pest in soybean fields, harbors a symbiont Burkholderia in a specialized posterior midgut region of insects. Every generation of second nymphs acquires new Burkholderia cells from the environment. We compared in vitro cultured Burkholderia with newly in vivo colonized Burkholderia in the host midgut using biochemical approaches. The bacterial cell envelope of in vitro cultured and in vivo Burkholderia differed in structure, as in vivo bacteria lacked lipopolysaccharide (LPS) O-antigen. The LPS O-antigen deficient bacteria had a reduced colonization rate in the host midgut compared with that of the wild-type Burkholderia. To determine why LPS O-antigen-deficient bacteria are less able to colonize the host midgut, we examined in vitro survival rates of three LPS O-antigen-deficient Burkholderia mutants and lysates of five different midgut regions. The LPS O-antigen-deficient mutants were highly susceptible when cultured with the lysate of a specific first midgut region (M1), indicating that the M1 lysate contains unidentified substance(s) capable of killing LPS O-antigen-deficient mutants. We identified a 17 kDa protein from the M1 lysate, which was enriched in the active fractions. The N-terminal sequence of the protein was determined to be a soybean Kunitz-type trypsin inhibitor. These data suggest that the 17 kDa protein, which was originated from a main soybean source of the R. pedestris host, has antibacterial activity against the LPS O-antigen deficient (rough-type) Burkholderia.

A de novo transcriptomic analysis to reveal functional genes in Apolygus lucorum.

The widespread planting of genetically engineered cotton producing the Cry1Ac toxin has led to significantly reduced pesticide applications since 1997. However, consequently, the number of green mirid bugs (GMB), Apolygus lucorum (Meyer-Dür) has increased. So far the GMB, instead of the cotton bollworm Helicoverpa armigera (Hübner), has become the major pest in the transgenic Bt cotton field and has influenced cotton yield. Disproportionately, only a few studies on GMB at molecular level have been reported. Libraries from both third instar nymphs and adults were sequenced using Illumina technology, producing more than 106 million short reads and assembled into 63 029 unigenes of mean length 597 nt and N50 813 nt, ranging from 300 nt to 9771 nt. BLASTx analysis against Nr, Swissprot, GO and COG was performed to annotate these unigenes. As a result, 26 478 unigenes (42.01%) matched to known proteins and 107 immune-related, 320 digestive-related and 53 metamorphosis-related genes were detected in these annotated unigenes. Additionally, we profiled gene expression using mapping based differentially expressed genes (DEGs) strategy between the two developmental stages: nymph and adult. The results demonstrated that thousands of genes were significantly differentially expressed at different developmental stages. The transcriptome and gene expression data provided comprehensive and global gene resources of GMB. This transcriptome would improve our understanding of the molecular mechanisms of various underlying biological characteristics, including development, digestion and immunity in GMB. Therefore, these findings could help elucidate the intrinsic factors of the GMB resurgence, offering novel pest management targets for future transgenic cotton breeding.

A specific cathepsin-L-like protease purified from an insect midgut shows antibacterial activity against gut symbiotic bacteria.

Because gut symbiotic bacteria affect host biology, host insects are expected to evolve some mechanisms for regulating symbiont population. The bean bug, Riptortus pedestris, harbors the Burkholderia genus as a gut symbiont in the midgut organ, designated as the M4 region. Recently, we demonstrated that the lysate of M4B, the region adjacent to M4, harbors potent antibacterial activity against symbiotic Burkholderia but not to cultured Burkholderia. However, the bona fide substance responsible for observed antibacterial activity was not identified in the previous study. Here, we report that cathepsin-L-like protease purified from the lysate of M4B showed strong antibacterial activity against symbiotic Burkholderia but not the cultured Burkholderia. To further confirm this activity, recombinant cathepsin-L-like protease expressed in Escherichia coli also showed antibacterial activity against symbiotic Burkholderia. These results suggest that cathepsin-L-like protease purified from the M4B region plays a critical role in controlling the population of the Burkholderia gut symbiont.

Burkholderia gut symbionts enhance the innate immunity of host Riptortus pedestris.

The relation between gut symbiosis and immunity has been reported in various animal model studies. Here, we corroborate the effect of gut symbiont to host immunity using the bean bug model. The bean bug, Riptortus pedestris, is a useful gut symbiosis model due to the monospecific gut symbiont, genus Burkholderia. To examine the effect of gut symbiosis to host immunity, we generated the gut symbiont-harboring (symbiotic) insect line and the gut symbiont-lacking (aposymbiotic) insect line. Upon bacterial challenges, the symbiotic Riptortus exhibited better survival than aposymbiotic Riptortus. When cellular immunity was inhibited, the symbiotic Riptortus still survived better than aposymbioic Riptortus, suggesting stronger humoral immunity. The molecular basis of the strong humoral immunity was further confirmed by the increase of hemolymph antimicrobial activity and antimicrobial peptide expression in the symbiotic insects. Taken together, our data clearly demonstrate that Burkhoderia gut symbiont positively affect the Riptortus systemic immunity.

Insect Gut Symbiont Susceptibility to Host Antimicrobial Peptides Caused by Alteration of the Bacterial Cell Envelope.

The molecular characterization of symbionts is pivotal for understanding the cross-talk between symbionts and hosts. In addition to valuable knowledge obtained from symbiont genomic studies, the biochemical characterization of symbionts is important to fully understand symbiotic interactions. The bean bug (Riptortus pedestris) has been recognized as a useful experimental insect gut symbiosis model system because of its cultivatable Burkholderia symbionts. This system is greatly advantageous because it allows the acquisition of a large quantity of homogeneous symbionts from the host midgut. Using these naïve gut symbionts, it is possible to directly compare in vivo symbiotic cells with in vitro cultured cells using biochemical approaches. With the goal of understanding molecular changes that occur in Burkholderia cells as they adapt to the Riptortus gut environment, we first elucidated that symbiotic Burkholderia cells are highly susceptible to purified Riptortus antimicrobial peptides. In search of the mechanisms of the increased immunosusceptibility of symbionts, we found striking differences in cell envelope structures between cultured and symbiotic Burkholderia cells. The bacterial lipopolysaccharide O antigen was absent from symbiotic cells examined by gel electrophoretic and mass spectrometric analyses, and their membranes were more sensitive to detergent lysis. These changes in the cell envelope were responsible for the increased susceptibility of the Burkholderia symbionts to host innate immunity. Our results suggest that the symbiotic interactions between the Riptortus host and Burkholderia gut symbionts induce bacterial cell envelope changes to achieve successful gut symbiosis.

Unveiling chemical defense in the rice stalk stink bug against the entomopathogenic fungus Metarhizium anisopliae.

Eggs, nymphs (1st-5th instar) and adults of Tibraca limbativentris were challenged by conidial suspensions of its major fungal pathogen Metarhizium anisopliae in order to assess their susceptibility. The role of chemical defensive compounds from exocrine secretions produced by both nymphs and adults were examined for their participation on M. anisopliae infection. Although insect susceptibility to M. anisopliae followed a dose-dependent manner, adults followed by older nymphs displayed the highest resistance. Eggs were highly susceptible showing >96% fungal infection. Crude extracts isolated from metathoracic scent gland and dorsal abdominal glands of adults and nymphs, respectively, showed fungistatic effects by impairing spore germination, vegetative growth and sporulation. Gas chromatography-mass spectrometry analysis of these extracts revealed that the major components were short-chain hydrocarbons (C10-13) and unsaturated aldehydes. In vitro tests with the corresponding synthetic standards indicated compounds with greater antifungal activity including (E)-2-hexenal, (E)-2-octenal, and (E)-2-decenal, with the latter being the most deleterious to fungal fitness. We demonstrated that differential susceptibility of the rice stalk stink bug to M. anisopliae infection is age-specific and partly mediated by fungistatic properties of aldehydes, which are produced by scent glands of both nymphs and adults.

The immune response of hemocytes of the insect Oncopeltus fasciatus against the flagellate Phytomonas serpens.

The genus Phytomonas includes parasites that are etiological agents of important plant diseases, especially in Central and South America. These parasites are transmitted to plants via the bite of an infected phytophagous hemipteran. Despite the economic impact of these parasites, many basic questions regarding the genus Phytomonas remain unanswered, such as the mechanism by which the parasites cope with the immune response of the insect vector. In this report, using a model of systemic infection, we describe the function of Oncopeltus fasciatus hemocytes in the immune response towards the tomato parasite Phytomonas serpens. Hemocytes respond to infection by trapping parasites in nodular structures and phagocytizing the parasites. In electron microscopy of hemocytes, parasites were located inside vacuoles, which appear fused with lysosomes. The parasites reached the O. fasciatus salivary glands at least six hours post-infection. After 72 hours post-infection, many parasites were attached to the salivary gland outer surface. Thus, the cellular responses did not kill all the parasites.

Effects of Artemisia annua L. (Asteracea) on the digestive enzymatic profiles and the cellular immune reactions of the Sunn pest, Eurygaster integriceps (Heteroptera: Scutellaridae), against Beauveria bassiana.

Plant extracts are currently studied more and more because of the possibility of their usage in plant protection. Many of the natural plant compounds which are used in the control of pests are known to affect the digestion and immune functions of insects. In this study, effects of Artemisia annua extract on the digestive enzymatic profiles and the cellular immune reactions of Eurygaster integriceps were investigated to reach a better understanding of its role in the control of this pest as the most destructive one in the production of wheat in the Near and Middle East, eastern and southern Europe and North Africa. Feeding and injection methods were used to study the plant extract effects on digestive enzymes and cellular immunity, respectively. When adult E. integriceps fed on food and water containing plant extracts, activity of the digestive enzymes, including alpha-amylase, alpha- and beta-glucosidases, protease and lipase, in addition to cellular immune reactions (total and differentiate hemocyte numbers, phagocytosis, nodule formation and phenoloxidase activity) against Beauveria bassiana were affected and significantly decreased in comparison with controls, in that the clear dose-response relationships were established with respect to enzyme activities and immune reactions. A. annua extract had a significant effect on kinetic parameters (Vmax and Km) of digestive enzymes and phenoloxidase activity so that the presence of the plant extract decreased the value of Vmax and increased Km, causing the reduction of enzyme affinity to the substrate, overall velocity of the reaction and finally interfering with the rate of breakdown of the enzyme-substrate complex. The understanding of fungal-induced immune responses and identification of factors regarding fungal virulence could be important in accelerating host death in a biological control scenario. Hence, the combination of botanical pesticides and microbes to control insect pest populations would be a safe and possibly rapid method to decrease their damage and environmental risk due to the use of chemical pesticides.

Blockades of phospholipase A(2) and platelet-activating factor receptors reduce the hemocyte phagocytosis in Rhodnius prolixus: in vitro experiments.

The hemocytes phagocytosis in response to microorganisms may play an important role in the cellular immune responses of insects. Here, we have evaluated the effects of the platelet-activating factor (PAF) and eicosanoids in the phagocytosis of hemocyte monolayers of Rhodnius prolixus to the yeast Saccharomyces cerevisiae. Experiments showed that the phagocytosis of yeast cells by Rhodnius hemocytes is very efficient in both controls and cells treated with PAF and arachidonic acid. Phagocytosis of yeast particles is significantly blocked when the specific phopholipase A(2) inhibitor, dexamethasone, is applied on the hemocytes. By contrast, dexamethasone-pretreated hemocyte monolayers exhibit a drastic increase in the quantity of yeast cell-hemocyte internalization when the cells are treated by arachidonic acid. In addition, phagocytosis presents significant reduction in hemocyte monolayers treated with a specific PAF receptor antagonist, WEB 2086. Nevertheless, inhibition of phagocytosis with WEB 2086 is counteracted by the treatment of the hemocyte monolayers with PAF. In conclusion, phagocytosis of yeast cells by hemocytes is related to the activation of PAF receptors and eicosanoid pathways in the bloodsucking bug, R. prolixus.

Occupational asthma due to grain pests Eurygaster and Ephestia.

BACKGROUND: Workers occupationally exposed to grain dust have a high prevalence of asthma. The pathogenesis of their respiratory symptoms remains obscure when sensitization to cereal allergens has not been proved. Given the ubiquity of arthropods in stored vegetable products, we have studied the allergenic potential of two very prevalent grain pests, Eurygaster and Ephestia, as a cause of occupational asthma. We have also studied the allergenic relationship between Anisakis simplex (AS) and these pests. METHODS: We selected 15 asthmatic workers exposed to cereal dust, in whom sensitization to cereal allergens was not clear. As controls, we selected a patient who suffered from anaphylaxis after the ingestion of cereals, 6 patients sensitized to different arthropods, 1 patient who suffered from asthma after inhaling fish flour contaminated with AS, and a pool of 40 asthmatic patients with different ethiologies not due to arthropods or cereals. We performed prick tests with these pests, AS, and pure and parasitized flours, bronchial challenges, specific IgE determination, and RAST inhibition. RESULTS: All of the 15 cereal-exposed workers were sensitized to Eurygaster and Ephestia. Only 2 had detectable levels of serum IgE to these pests, but 13 presented a positive prick test result and IgE to AS without problems after eating fish. Bronchial challenges were positive to Eurygaster in 7 patients and to Ephestia in 2 patients. Twelve patients had positive prick tests to parasitized flour but not to pure flour. The patient with cereal anaplylaxis presented sensitization to both flours but not to AS. In the control group, the patient who suffered from asthma after the inhalation of AS-parasitized fish flour presented a positive prick test result and high levels of specific IgE to these pests. The RAST inhibition showed cross-reactivity between Eurygaster and AS in this patient, but the inhibition was not as evident with the sera of other workers. The same tests in the group of 40 asthmatic patients were negative. CONCLUSIONS: The clinical importance of pests as a cause of baker's asthma should not be underestimated. Sensitization to Eurygaster and Ephestia could be important in the asthma episodes suffered by our patients, and the parasitized wheat was found to have a higher allergenic potential than pure cereal flour.