Roger F. Hou

Immunolocalization of Prophenoloxidase in the Process of Wound Healing in the Mosquito Armigeres subalbatus (Diptera: Culicidae)

Abstract 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.

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.

Effects of inhibitors of serine protease, phenoloxidase and dopa decarboxylase on the melanization of Dirofilaria immitis microfilariae with Armigeres subalbatus haemolymph in vitro

The melanization of Dirofilaria immitis microfilariae in Armigeres subalbatus haemolymph in vitro is a two-step process. Firstly, the microfilariae are encased in a transparent capsule, then the capsule material is melanized later. Benzamadine HC1 and p-amidinophenylmethanesulfonyl fluoride, both serine protease inhibitors, inhibited the deposition of the transparent capsule material and melanization. Diethyldithiocarbamate, a phenoloxidase inhibitor, did not prevent the deposition of the transparent capsule material but it did inhibit melanization. m-Hydroxybenzylhydrazine, a dopa decarboxylase inhibitor, had no inhibitory action on the deposition of the transparent capsule material. However, the time for the transparent capsule to become melanized was delayed due to the presence of m-hydroxybenzylhydrazine in the test system. Immmunocytochemical localization showed that propheoloxidase and/or phenoloxidase was present in the transparent capsule material. These results suggest (1) that prophenoloxidase cascade is essential in the melanization of microfilariae in mosquitoes and its putative activation behaviour is similar to that of other insects and (2) that the deposition of transparent capsule material is the result of the activation of prophenoloxidase by serine proteases and the melanization of the transparent capsule material is due to the presence of active phenoloxidase in capsule.

Physiological roles of a yeast-like symbiote in reproduction and embryonic development of the brown planthopper, Nilaparvata lugens Stål

Abstract The physiological roles of a yeast-like symbiote in oviposition and embryonic development of its host, the brown planthopper, Nilaparvata lugens Stal, were studied using heat treatment, lysozyme injection and ligation of eggs. The eggs laid by the heat-treated females harboured only a few of the symbiotes, and their symbiote ball through embryonic development was free of symbiotes. The embryos of subsymbiotic eggs could not undergo blastokinesis and dorsal closure, and failed to hatch due to lack of differentiation of the abdominal segments. Electrophoretic profile of the eggs laid by the heat-treated females indicated the absence of several minor proteins which are usually found in the fat body of normal females. A protein (Y) of 131 kD was barely detectable in the heat-treated insects, and could not be found in the ligated eggs in which the symbiote ball was completely separated from the developing germ band. It is suggested that the symbiote supplies its host with proteins for normal embryonic and postembryonic development. The number of yeast-like symbiotes in female adults was reduced after injection with lysozyme solution, and some of the eggs were unable to hatch due to failure in blastokinesis; this was similar to the heat-treated insects. The embryos of ligated eggs could complete segmentation and differentiation normally before 110 h, but the abdominal segments failed to differentiate after dorsal closure, and regressed leaving only the head. Partially ligated eggs harboured some symbiotes and could produce normal larvae. It is concluded that the yeast-like symbiote is significant in abdominal segmentation and differentiation of the planthopper embryo.

Deterrent effect of the chinaberry extract on oviposition of the diamondback moth, Plutella xylostella (L.) (Lep., Yponomeutidae)

Abstract: Oviposition of the diamondback moth, Plutella xylostella (L.), was deterred by extracts from fruits of the chinaberry, Melia azedarach L., grown in Taiwan. The oviposition was significantly reduced by 49.6, 86.6 and 93.5% in choice tests and 46.2, 72.1 and 80.2% in no‐choice tests at concentrations of 0.5, 2.0 and 4.0%, respectively. In addition to ovipositional deterrence, chinaberry fruit extract showed repellency to the moth when applied to rape seedlings.

Effects of chinaberry fruit extract on feeding, growth and fecundity of the diamondback moth, Plutella xylostella L. (Lep., Yponomeutidae)

Abstract: Effects of chinaberry fruit extracts on larval mortality, feeding inhibition and reproduction of the diamondback moth (DBM), Plutella xylostella L., were investigated by feeding DBM larvae on treated leaves or seedlings. These extracts were found to be toxic to DBM larvae. The larvae usually died from failure in molting. The developmental growth rates and the food consumption were also reduced at concentrations of 2.0 and 4.0%. Chinaberry extracts reduced pupal weight, adult emergence and longevity in a dose‐dependent manner when newly hatched larvae were continuously reared on treated rape seedlings at concentrations of 0.05% or above. Fecundity of the resulting females from the larvae treated with 0.5% extract was also reduced, while the egg hatch was not affected. However, the extracts significantly decreased egg hatch when the eggs were dipped directly into test solutions at 1.0% or above.

Electron Microscopic Observations on Wound-Healing in Larvae of the Mosquito Armigeres subalbatus (Diptera: Culicidae)

Abstract The wound-healing processes in the mosquito Armigeres subalbatus (Coquillett) were observed with electron microscopy. The initial reaction involved wound contraction and aggregation of injured surface tissues, cell debris and movement of granulocytes toward the wound. Granulocytes first aggregated around the surface of the wound and many filamentous filopodia protruded to connect with cytoplasmic strands. These strands were then interconnected to form a network coagulum resulting in wound closure to prevent body fluid loss. Granulocytes lysed on the wound-site and released granular materials around the wound, inducing localized clot formation. These results suggested that wound-healing in this mosquito species involved both humoral and cellular reactions. The latter reaction involved the movement of plasmatocytes to the basement membrane of the epidermis beneath the wound-site and epithelial cells regeneration. Our observations revealed that wound-healing in A. subalbatus involves the wound contraction, formation of a temporary cellular clump, scar formation, basement membrane formation, and reepithelialization. The larvae neither discarded the wound scar nor secreted a new cuticle until the next molting. Based on the ultrastructural observations, it is suggested that the wound-healing reaction in A. subalbatus was probably a typical response employed by other members of the family Culicidae.

Complete genome sequence and genetic organization of a Taiwan isolate of Zucchini yellow mosaic virus

The complete nucleotide sequence of the RNA genome of a Taiwan isolate of Zucchini yellow mosaic virus (ZYMV TW-TN3) was determined from five overlapping cDNA clones 9591 nucleotides in length excluding the poly (A) tail. Computer analysis of the sequence revealed a large open reading frame (ORF) that encodes a polyprotein of 3080 amino acids. Comparison of the gene products of TW-TN3 with those of the reported California (CA), Reunion Island (RU), and Singapore (S) isolates of ZYMV revealed that P1 protein is most variable, with amino acid identities of 59.0-93.2%. The 5′untranslated region (UTR) of TW-TN3 shares 61.6-83.3% nucleotide identities, and the 3′UTR shares 90.4-95.7% nucleotide identities, with those of the other isolates. A phylogenetic tree derived from the sequences of P1 proteins of TW-TN3 and the other six reported ZYMV isolates revealed four major genotypes. TW-TN3 was classified in genotype I, and US isolates were in genotype II. The Reunion Island and Singapore isolates were separated into genotypes III and IV, respectively. The distance relationships of P1 protein of genotype I were closer to genotype II, indicating that the Taiwan and US isolates may evolve from the same ancestor. Analyses on the cleavage sites of the C-terminal halves of the polyproteins of TW-TN3, CA, RU, and S isolates revealed that NIa protease cleaves at Q-S, and E-S dipeptide sequences, with a consensus sequence of V-x-x-(Q, E)/(S, A, G). The genetic organization of TW-TN3 was concluded as Vpg/5′ leader/P1 (36kDa)/HC Pro (52kDa)/P3 (40kDa)/6Kl (6kDa)/CIP (71kDa)/6K2 (6kDa)/NIa-Vpg (22kDa)/NIa-Pro (27kDa)/NIb (60kDa)/CP (31kDa)/3′ UTR-poly(A) tract.

Continuous rearing of the aster leafhopper, Macrosteles fascifrons, on a chemically defined diet

Abstract A holidic diet for feeding the aster leafhopper, Macrosteles fascifrons , was formulated. The amino acids, B-vitamins, and sucrose are less concentrated than in aphid diets. Cholesterol, at 5 mg/ml, is required for the last ecdysis. Although leafhoppers reared on this diet have poorer survival and shorter life span than those reared on plants, they produce more progeny. Leafhoppers reared on this diet have completed the ninth generation and the culture is still thriving.

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.