The Invasion and Encapsulation of the Entomopathogenic Nematode, Steinernema abbasi, in Aedes albopictus (Diptera: Culicidae) Larvae.

The Asian tiger mosquito, Aedes albopictus, is of crucial concern to the public and veterinary health because of its vector role in transmission of several mosquito-borne diseases. Over the past decades, entomopathogenic nematodes (EPNs) have been used to control important agricultural insect pests and are considered to be effective against mosquitoes as well. The objectives of this study were to investigate the mosquitocidal effects of Steinernema abbasi to Ae. albopictus and the encapsulation processes of invading nematodes in the mosquito host. In this study, we found that S. abbasi was pathogenic to 3rd and 4th instar larvae of Ae. albopictus by entering the hemocoel of the 3rd and 4th instar larvae mainly through mouth and gastric caecum or by penetrating pupae through the intersegmental membrane or trumpet. The mosquito larvae infected with a single nematode caused a high mortality. Although EPNs in the hemocoel of mosquitoes were melanized and encapsulated, most Ae. albopictus larvae failed to survive after infection with S. abbasi. Overall, we demonstrated that S. abbasi is pathogenic to Ae. albopictus larvae, suggesting that this S. abbasi isolate has potential as a biocontrol agent for managing this vector mosquito.

Steinernema taiwanensis n. sp. (Rhabditida: Steinernematidae), a new entomopathogenic nematode from Taiwan.

A new isolate of the entomopathogenic nematode, Steinernema taiwanensis n. sp., was isolated from soil in Pingtung County, Taiwan. This new species could be characterized and distinguished from other related species by its morphological characters, morphometrics, and phylogenetic analysis. The body length and distance from anterior end to nerve ring of infective juveniles is 1012 (983-1045) µm and 124 (120-127) µm, lateral field formula 2, 6, 7, 8, 2, and the tail length 90 (79-96) µm without dorsal constriction in tail region. The first generation males of S. taiwanensis n. sp. are characterized by spicule shape, smooth blade tip, 23 genital papillae (11 pairs and 1 single papilla), spicule length of 94 (89-99) µm and gubernaculum length of 68 (65-70) µm. Females from the first generation of S. taiwanensis n. sp. have no epiptygmata and a slightly developed post-anal swelling. Phylogenetic analysis of ITS and D2D3 regions of rDNA showed that S. taiwanensis n. sp. belongs to the Longicaudum-clade and comprises a monophyletic group with S. guangdongense and S. longicaudum. The new isolate is described as a novel species according to morphological and phylogenetic analyses.

In vitro activity of mastoparan-AF alone and in combination with clinically used antibiotics against multiple-antibiotic-resistant Escherichia coli isolates from animals.

The in vitro activity of mastoparan-AF, an amphipathic antimicrobial peptide isolated from the hornet venom of Vespa affinis, alone and in combination with various clinically used antibiotics, was investigated against 21 Escherichia coli isolates/strains. Most E. coli isolates tested were detected containing multiple-antimicrobial resistance genes. Antimicrobial activity of mastoparan-AF was measured by MIC, MBC, time-kill kinetic assay and chequerboard titration method. Mastoparan-AF exhibited potent antimicrobial activity against most multiple-antibiotic-resistant E. coli isolates at the concentrations ranging from 4 to 16 µg/ml. Combination studies showed that mastoparan-AF acts synergistically with certain antibiotics, i.e., cephalothin or gentamicin, against some multiple-antibiotic-resistant E. coli isolates. In conclusion, mastoparan-AF alone or in combination with other antibiotics could be promising as alternatives for combating multiple-antibiotic-resistant E. coli in future clinical applications.

The bacterium associated with the entomopathogenic nematode Steinernema abbasi (Nematoda: Steinernematidae) isolated from Taiwan.

A symbiotic bacterium of the entomopathogenic nematode, Steinernema abbasi, isolated from Taiwan, determined to be a species of Xenorhabdus based on its physiological and biochemical characteristics has been determined to be similar to Xenorhabdus indica of S. abbasi Oman isolate as based on sequence analyses of 16S rDNA.

Effectiveness of attract-and-kill systems using methyl eugenol incorporated with neonicotinoid insecticides against the oriental fruit fly (Diptera: Tephritidae).

Laboratory bioassays and field trials were conducted to evaluate an "attract-and-kill" system using methyl eugenol (ME) with neonicotinoid insecticides against male oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae). In laboratory bioassays, mortality of male flies resulting from the conventional toxicant, naled was 98.3-100% at 24 through 72 h after treatment, whereas the neonicotinoid insecticides imidacloprid and acetamiprid caused only approximately 60-80% at 24 through 72 h after treatment. In the assays of residual effect, naled was persistent up to 96 wk, whereas imidacloprid or acetamiprid was persistent up to 150 wk, resulting in 38.9 or 61.2% male mortality, respectively. Imidacloprid, in particular, caused a delayed lethal effect on flies. In another experiment, male mortality within 28 wk from clothianidin, another neonicotinoid insecticide, was approximately 80% after exposure for 24 h, suggesting a delayed lethal effect similar to those treated with imidacloprid, and mortality was up to 91.8%, if observed, 72 h after treatment. In field trials, attractiveness was similar between ME alone and ME incorporated with naled or neonicotinoids, indicating that addition of these insecticides to ME in traps is not repellent to B. dorsalis males. Using an improved wick-typed trap with longer attractiveness for simulating field application, addition of imidacloprid or acetamiprid maintained 40.1 or 64.3% male mortality, respectively, when assayed once every 2 wk from traps placed in orchards for 42 wk without changing the poison, whereas incorporation with naled resulted in as high as 98.1% after 34 wk and approximately 80% at 42 wk, indicating that persistence is increased compared with sugarcane fiberboard blocks for carrying poison attractants. This study also suggests that neonicotinoid insecticides could be used as an alternative for broad-spectrum insecticides as toxicants in fly traps.

Induction of apoptosis in SF21 cell line by conditioned medium of the entomopathogenic fungus, Nomuraea rileyi, through Sf-caspase-1 signaling pathway.

The apoptosis in SF-21 cell line can be induced by the conditioned medium (CM) of the entomopathogenic fungus, Nomuraea rileyi, based on changes in morphology and formation of apoptotic bodies in cultured cells, and with the onset of DNA fragmentation as shown by TUNEL staining and agarose electrophoresis. Moreover, the induction of apoptosis in SF-21 cells was inhibited by adding the inhibitor of effector caspase, viz. z-DEVD-fmk, to the CM, indicating that Sf-caspase-1 is involved in this apoptosis. Similarly, the inhibitor of initiator caspase, viz., z-VAD-fmk, inhibited apoptosis. Therefore, both initiator and effector caspases are possibly involved in the apoptosis of SF-21 cells. In addition, we detected Sf-caspase-1 activity in the process of apoptosis in SF-21 cells, suggesting that the effector caspase in SF-21 is similar to that found in mammalian cells. Our results also indicated that the apoptosis found in this line is accomplished through a Sf-caspase-1 signaling pathway.

Modifications of the Helper Component-Protease of Zucchini yellow mosaic virus for Generation of Attenuated Mutants for Cross Protection Against Severe Infection.

ABSTRACT A nonpathogenic mild strain is essential for control of plant viruses by cross protection. Three amino acid changes, Arg(180)-->Ile(180) (GA mutation), Phe(205)-->Leu(205) (GB mutation), and Glu(396)-->Asn(396) (GC mutation), of the conserved motifs of the helper component-protease (HC-Pro) of a severe strain TW-TN3 of Zucchini yellow mosaic virus (ZYMV), a member of the genus Potyvirus, were generated from an infectious cDNA clone that carried a green fluorescent protein reporter. The infectivity of individual mutants containing single, double, or triple mutations was assayed on local and systemic hosts. On Chenopodium quinoa plants, the GB mutant induced necrotic lesions; the GA, GC, and GBC mutants induced chlorotic spots; and the GAB and GAC mutants induced local infection only visualized by fluorescence microscopy. On squash plants, the GA, GB, GC, and GBC mutants caused milder mosaic; the GAC mutant induced slight leaf mottling followed by recovering; and the GAB mutant did not induce conspicuous symptoms. Also, the GAC mutant, but not the GAB mutant, conferred complete cross protection against the parental virus carrying a mite allergen as a reporter. When tested on transgene-silenced transgenic squash, the ability of posttranscriptional gene silencing suppression of the mutated HC-Pro of GAC was not significantly affected. We concluded that the mutations of the HC-Pro of ZYMV reduce the degrees of pathogenicity on squash and also abolish the ability for eliciting the hypersensitive reaction on C. quinoa, and that the mutant GAC is a useful mild strain for cross protection.

Restriction of vaccinia virus replication by a ced-3 and ced-4-dependent pathway in Caenorhabditis elegans.

Genetic tractability and easy manipulation make Caenorhabditis elegans a good model to study host-pathogen interactions. Dozens of different bacterial species can pathogenically infect C. elegans under laboratory conditions, and all of these microbes are extracellular pathogens to nematodes. Viruses, on the other hand, are obligate intracellular parasites, and yet no viral infections have been reported for C. elegans. We established a procedure allowing vaccinia virus to enter and subsequently replicate in C. elegans. Virus replication was significantly enhanced in ced-3, ced-4, ced-9(gf), and egl-1(lf) mutants, demonstrating that the core programmed cell death (PCD) genes ced-3, ced-4, ced-9, and egl-1 control vaccinia virus replication in C. elegans. The ability of ced-3 and ced-4 alleles to restrict virus replication is correlated with their cell-killing activities. Moreover, the increase in vaccinia virus replication levels in the PCD-defective mutants was not likely to be caused by the extra live cells, as neither the inhibition of PCD by icd-1 overexpression nor the presence of extra cells after extra cell divisions in cul-1 or lin-23 mutants had any significant effect on vaccinia virus replication. Therefore, the core PCD genes possess a unique function in controlling vaccinia virus replication in C. elegans.

Determination and distribution of a female-specific protein in the brown planthopper, Nilaparvata lugens Stal (Homoptera: Delphacidae).

Electrophoretic analyses of hemolymph and body or ovary homogenates from reproducing females, males, and 5th instar nymphs of the brown planthopper, Nilaparvata lugens Stal, revealed a protein band of 175 kDa in females. An immunoblot test using antibody against this protein showed a positive reaction with a 175 kDa protein from female body or ovary homogenates. It is likely that this protein in hemolymph is vitellogenin (Vg). Distribution of Vg was determined by immunofluorescence and immunogold labeling techniques. The results showed that the positive immunofluorescence reactions were present in yolk particles, the intercellular space of follicle cells, hemolymph, and the epithelial plug of ovarioles. In addition, the yeast-like symbiotes (YLS) in mycetocytes of adults and various nymphal instars as well as those free in hemolymph or entering oocytes also exhibited a positive reaction. Electron micrographs showed that immunogold particles were found most in yolk mass and YLS over other tissues. Especially the YLS in various developmental stages all contained immunogold particles, implying that the symbiote is somewhat related with production of the female-specific protein.

Elevation of plasminogen activators in cerebrospinal fluid of mice with eosinophilic meningitis caused by Angiostrongylus cantonensis.

A hallmark of parasitic meningitis is the infiltration of eosinophils into the subarachnoid space. Infection with Angiostrongylus cantonensis in mice induced proteinase activity in parallel with the pathological changes of eosinophilic meningitis. Zymogram analysis demonstrated that 70 and 55 kDa proteinases from cerebrospinal fluid (CSF) were active against the casein/plasminogen substrate. The proteinase activities were clearly inhibited by phenylmethanesulphonyl fluoride but not by ethylenediamine tetraacetic acid, 1,10-phenanthroline or leupeptin. Western blotting confirmed these enzymes to be tissue-type plasminogen activator and urokinase-type plasminogen activator, respectively. High activities of tissue-type plasminogen activator and urokinase-type plasminogen activator were detected in the CSF of mice with eosinophilic meningitis, and correlated positively with CSF eosinophil numbers and total protein, respectively. Immunohistochemistry demonstrated that tissue-type plasminogen activator and urokinase-type plasminogen activator localised in the endothelial cells of blood vessels, in blood clots and in infiltrated leukocytes. These results suggest that tissue-type plasminogen activator and urokinase-type plasminogen activator may be play a role in the pathogenesis of eosinophilic meningitis of angiostrongyliasis.

Immunolocalization of prophenoloxidase in the process of wound healing in the mosquito Armigeres subalbatus (Diptera: Culicidae).

Hemolymph coagulation began almost immediately after wounding in mosquito, Armigeres subalbatus, (Coquillett) larvae. Immunocytochemical localization showed that prophenoloxidase (pro-PO) was distributed in the wound site. In the initial wounding, coagulation and wound plug formation occurred with granulocyte migration. The hemocytes lysed and released granular materials around the wound site, prophenoloxidase being mostly localized in granules and cuticle. In the second phase of wound healing, melanin accumulation occurred at the wound site along the margin of the cuticle and rapidly increased in thickness. Immunogold-labeled pro-PO was localized in vacuoles, melanins, and cuticle, with the gold particles labeled intensely on the undarkened cuticle and weakly on the darkened cuticle. It is believed that pro-PO is activated upon wound initiation to produce melanin product and deposited on the cuticle. In the final phase of healing, scab melanization and pro-PO immunogold localization were reduced and accompanied by epithelial cell regeneration. This proenzyme was localized in vesicles and flocculent materials, but was absent in the melanized scab. Our results further indicate that pro-PO was present in granules, cuticles, epithelial cells, vacuoles, and flocculent materials but not in melanized scab and coagulated clot. The pro-PO immunogold particles labeled intensely in the initial wounding but weakly in the final phase. Our observations also suggest that pro-PO is released from granulocytes by cell rupture, synthesized or stored in granulocytes, and then is released into the wound site via the cytoplasmic granules. This study indicates that the pro-PO is involved in numerous physiological roles in the process of wound healing in this mosquito.