Phagocytosis and immune response studies of Macrophage-Nanodiamond Interactions in vitro and in vivo.

The applications of nanodiamond as drug delivery and bio-imaging can require the relinquishing ND-drug conjugate via blood flow, where interaction with immune cells may occur. In this work, we investigated the ND penetration in macrophage and the immune response using the tissue-resident murine macrophages (RAW 264.7). Confocal fluorescence imaging, immunofluorescence analysis of nuclear translocation of interferon regulatory factor IRF-3 and transcriptional factor NF-κΒ, analysis of pro-inflammatory cytokines production IL-1ß, IL-6 IL-10 with a reverse transcription-polymerase chain reaction technique were applied. The TNF-α factor production has been studied both in vitro at ND interaction with the macrophage and in vivo after ND injection in the mice blood system using immunoassay. The macrophage antibacterial function was estimated through E. coli bacterial colony formation. ND didn't stimulate the immune response and functionality of the macrophage was not altered. Using MTT test, ND was found negligibly cytotoxic to macrophages. Thus, ND can serve as a biocompatible platform for bio-medical applications. Left: Graphic representation of Nanodiamond internalization in macrophage. Right: (a) Fluorescence images of lysosomes, (b) nanodiamond and (c) merged image of nanodiamond internalization in macrophage.

Inhibition of group A streptococcal infection by Melaleuca alternifolia (tea tree) oil concentrate in the murine model.

AIMS: To investigate the effect of a water-soluble Melaleuca alternifolia concentrate (MAC) on group A streptococcus (GAS; Streptococcus pyogenes)-induced necrotizing fasciitis. METHODS AND RESULTS: MAC pretreatment (1% and 2% v/v) was able to protect mice from GAS infection in an air pouch model. GAS-induced mouse death and skin injury were inhibited dose dependently by MAC. Administration of MAC at 6 h post-GAS infection partially delayed mouse death. Surveys of the exudates of the air pouch of MAC-treated mice revealed that the survival of infiltrating cells was prolonged, the bacteria were eliminated, and the production of inflammatory cytokines was inhibited. MAC could directly inhibit the growth of GAS in vitro, and the minimal inhibitory concentration (MIC) of MAC for GAS was determined as 0.05% v/v using the time-kill assay. Furthermore, a sub-MIC dose of MAC not only enhanced the bactericidal activity of RAW264.7 macrophage cells against GAS but also increased susceptibility of GAS for blood clearance. CONCLUSIONS: These results suggest that MAC may inhibit GAS-induced skin damage and mouse death by directly inhibiting GAS growth and enhancing the bactericidal activity of macrophages. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results provide scientific data on the use of MAC for the treatment of GAS-induced necrotizing fasciitis in the murine model.

Streptococcal pyrogenic exotoxin B antibodies in a mouse model of glomerulonephritis.

Streptococcal pyrogenic exotoxin B is an extracellular cysteine protease. Only nephritis-associated strains of group A streptococci secrete this protease and this may be involved in the pathogenesis of post-streptococcal glomerulonephritis. Mice were actively immunized with a recombinant protease inactive exotoxin B mutant or passively immunized with exotoxin B antibody. Characteristics of glomerulonephritis were measured using histology, immunoglobulin deposition, complement activation, cell infiltration, and proteinuria. None of the mice given bovine serum albumin or exotoxin A as controls showed any marked changes. Immunoglobulin deposition, complement activation, and leukocyte infiltration occurred only in the glomeruli of exotoxin B-hyperimmunized mice. One particular anti-exotoxin B monoclonal antibody, 10G, was cross-reactive with kidney endothelial cells and it caused kidney injury and proteinuria when infused into mice. This cross-reactivity may be involved in the pathogenesis of glomerulonephritis following group A streptococcal infection.

A new cell-based assay for measuring the forward mutation rate of HIV-1.

Over 20 years into the ever-worsening AIDS pandemic, genetic variation remains the greatest obstacle for treating and preventing HIV-1 infection. Mutation rate assays for HIV-1 have been reported; however, none measure directly the forward mutation rate during replication of the virus in cell culture while still retaining the ability to propagate and further study mutant proviruses. Therefore, the objective of the current study was to develop such a phenotypic cell-based assay for measuring the forward mutation rate of HIV-1. Conventional recombinant DNA techniques and polymerase chain reaction were used to create a replication defective HIV-1 vector, pNL4-3Delta+cass, which is based on the NL4-3 strain and contains the thymidine kinase gene from human herpes virus type 1 as the mutational target. A series of transfection and infection steps were used to introduce the vector into 143B cells, which are negative for thymidine kinase function, and produce vector virus for a single cycle of replication. Viral titers were measured by counting the number of drug resistant colonies on the assay plates, and forward mutation rates were calculated from the viral titers. Mutant proviruses were sequenced to determine the types of genetic alterations that occurred. The average forward mutation rate for HIV-1 was 2.2 x 10(-5)mutations/base/cycle. The majority of mutations were base substitutions, including high frequencies of C-->U and G-->A transitions. Single adenosine insertions were also observed frequently. The new assay is economical and provides a direct measurement of the mutation rate during a single cycle of viral replication. Target cells containing mutant proviruses survive the drug selection process and may be propagated for further analysis. The new assay is novel and has many advantages over previous mutation rate assays and thus will be very useful in future studies on genetic variation of HIV-1.

Stability of desmopressin loaded in liposomes.

Desmopressin-containing liposome formulations have been developed for intranasal administration previously. Positively charged liposomes were found to be an efficient delivery system for desmopressin. In this study, stability of the loaded desmopressin in positively charged liposomes was further investigated. Comparison of the stability of desmopressin in solution and liposomes was made. Degradation of desmopressin was shown to follow a pseudo-first-order reaction. Degradation of desmopressin in both solution and liposomes demonstrated the same kinetic behavior and exhibited no significant difference in half-lives. Similar v-shape pH-rate profile was found for desmopressin degradation in solution and liposomes. At pH 4.0, the inflection point of the v-shape pH-rate curve, the reaction rate of desmopressin was lowest and the stability was greatest. The stability of lipid ingredients of dioleoylphosphatidylcholine (DOPC), cholesterol (C), and stearylamine (S) in the liposome dispersion at pH 4.0 was studied. Results demonstrated that DOPC, C, and S were relatively stable in the liposome structure when formulated with desmopressin. The degradation of desmopressin in solution and liposomes in the presence of alpha-chymotrypsin was investigated. A longer half-life for desmopressin in liposomes than in solution was observed. It was suggested that desmopressin was protected by the liposomes against alpha-chymotrypsin digestion.

Bovine ephemeral fever in Taiwan.

Bovine ephemeral fever (BEF) is a vector-borne disease of cattle, spanning tropical and subtropical zones of Asia, Australia, and Africa, caused by Ephemerovirus of the Rhabdoviridae. Taiwan has had 3 BEF epizootics, occurring in 1989, 1996, and 1999, since the vaccination regimen was initiated in 1984, given once a year in the spring with a single-dose formaldehyde-inactivated vaccine using the 1983 isolate as the seed virus. This study evaluated the 1999 population immunity against BEF virus in Taiwanese dairy cows with a neutralization test and whether the recent BEF virus isolates have mutated significantly from the vaccine virus. In March 1999, before vaccination, 94% of the animals studied were already seropositive, suggestive of an endemic or persistent infection from the previous year. By June 1999, when 51% of herds had been vaccinated, the antibody level rose, and by September 1999, the serum-neutralizing antibody (SNA) level fell to a minimum, preceding the outbreak of BEF in October 1999, during which the antibody levels of vaccinated cows continued to decline while those of unvaccinated cows rose sharply. The results suggest that, in 1999, vaccine-induced immunity was partially protective against BEE Because the current single-dose vaccination regimen resulted in minimal population immunity by September, a booster vaccination given in late summer may be advisable for future disease control. Analysis of the glycoprotein gene of Taiwanese isolates between 1983 and 1999 showed a 97.4-99.6% homology, with an alteration of 4 amino acids in antigenic sites G1, G3b, and G3c. Phylogenetic analysis of Taiwanese isolates revealed at least 2 distinct clusters: the 1983-1989 isolates and the 1996-1999 isolates. Both were distinct from 2 Japanese strains and the Australian BB7721 strain. Thus, at least 2 distinct BEF viruses, which had diverged before 1983, existed in Taiwanese dairy cows.

Preparation of desmopressin-containing liposomes for intranasal delivery.

The loading and leakage characteristics of the desmopressin-containing liposomes and the effect of liposomes on the nasal mucosa permeation and antidiuresis of desmopressin were investigated. Higher loading efficiency of desmopressin for positively charged liposomes than negatively charged liposomes was obtained, and neutral liposomes resulted in a similar loading efficiency as that of positively charged liposomes. Greater leakage of desmopressin from negatively charged liposomes than from positively charged and neutral liposomes was shown. The increase of permeability of desmopressin through the nasal mucosa indicated positively charged liposomes>negatively charged liposomes>solution. It was suggested that the enhanced contact time of positively charged liposomes with negatively charged nasal mucosa led to a high local desmopressin concentration on the penetration site to promote an effective penetration of desmopressin through the nasal mucosa. The desmopressin antidiuresis result after intranasal administration was in good agreement with the permeability result in the order of positively charged liposomes>negatively charged liposomes>solution. One of the mechanisms for the explanation of the best result on the enhancement of antidiuresis for positively charged liposomes may be the bioadhesive effect of the liposomes on the negatively charged nasal mucosa.

Enhancement of nasal absorption of calcitonin loaded in liposomes.

Intranasal administration of calcitonin-containing liposomes in rabbits was investigated to evaluate the in vivo calcitonin absorption performance. Plasma calcitonin concentrations and calcium levels were measured and pharmacokinetic parameters were calculated. The bioavailability of calcitonin resulted from the intranasal delivery formulations demonstrated an order of calcitonin-containing positively charged liposomes > calcitonin-containing negatively charged liposomes > calcitonin solution. The significant enhancement of bioavailability of calcitonin for positively charged liposomes may be due to the charge interaction of positively charged liposomes with the negatively charged mucosa surface. Marked accumulation of positively charged liposomes was found on the negatively charged nasal mucosa surface. The retention of positively charged liposomes on the nasal mucosa resulted in an increase of residence time with high local concentration of calcitonin for increase of absorption.

Acyclovir-containing liposomes for potential ocular delivery. Corneal penetration and absorption.

The in vitro corneal penetration and in vivo corneal absorption of acyclovir from an acyclovir-containing liposome system were investigated. Results of in vitro corneal penetration demonstrated that positively charged liposomes resulted in a penetration rate lower than those of negatively charged liposomes and free acyclovir in solution. An in vivo study indicated that the extent of acyclovir absorption from positively charged liposomes was higher that those from negatively charged liposomes and free acyclovir. The acyclovir concentration in the cornea after administration of positively charged liposomes showed that an acyclovir deposition in the cornea was greater than those of negatively charged liposomes and free acyclovir. From morphological observation of the cornea surface treated with liposomes, it was suggested that positively charged liposomes formed a completely coated layer on the cornea surface. These liposomes would bind intimately on the cornea surface, leading to an increase of residence time. Therefore, positively charged liposomes resulted in an increase of acyclovir (ACV) absorption.

Acute and subacute toxicity study of water-soluble polyalkylsulfonated C60 in rats.

Polyalkylsulfonated C60, or FC4S, a highly water-soluble caged fullerene derivative, is believed to be a free radical remover or an antioxidant in biological systems. A 50 mg/ml aqueous solution was prepared as a master solution and administered to female Sprague-Dawley CD(Crl:CD(SD)BR) rats in a single-dose acute toxicity study or a 12-day subacute toxicity study where rats were given the solution daily. In a study of the median lethal dose (LD50), no rats died after oral administration, and thus FC4S was considered to be nontoxic if administered orally. In an LD50 intraperitoneal injection study, rats died within 30 hr after injection; the LD50 was determined to be approximately 600 mg per kilogram of body weight. Rats injected with the compound intraperitoneally or intravenously immediately eliminated the compound through the kidney; the kidney appeared to be the primary target organ. The compound induced a distinct lysosome-overload nephrosis, a phagolysosomal nephropathy characterized by a tinctorial difference between the outer cortex and the inner cortex and the medulla. The affected outer cortex showed a diffuse degeneration, with the presence of numerous large vacuoles and cytoplasmic aggregates in the tubular epithelium. The phagolysosomal nephropathy was detected in rats after acute exposure as well as in the surviving rats following 1 intraperitoneal injection of 500 mg/kg or intravenous injection of 100 mg/kg. Ultrastructural investigation revealed numerous membranous conglomerates characteristic of phagolysosomal and/or lysosomal inclusions in the cytoplasm of the renal tubular epithelium. These conglomerates were confined to the vacuole, electron-dense, and unevenly stained. They varied in size and shape and were fused or aggregated. Occasional phagolysosomes were also observed in the endothelial cells of the peritubular plexus. A preliminary study of microsomal enzyme activity analysis revealed a suppression effect of liver cytochrome P-450-dependent monooxygenase activities, including cytochrome P-450, cytochrome b5, and benzo(a)pyrene hydroxylase, but an increased level of kidney cytochrome P-450-dependent monooxygenase activities, including NADPH-cytochrome P-450 reductase. The significance of these enzyme alterations was not well determined. Further study is needed to clarify the correlation between the alterations of microsomal enzyme activity and the nephropathy of lysosomal overload-induced changes. These changes may serve as a biological marker in toxicity screening tests for this class of compound.

Induction of thymocyte apoptosis in mice by Yersinia enterocolitica products.

In-vivo administration of the culture supernates from Yersinia enterocolitica resulted in thymus atrophy in C3H/HeJ mice, known to be lipopolysaccharide (LPS)-nonresponders. The thymocytes underwent apoptosis as characterised by fragmented DNA ladders on agarose gel electrophoresis, a cell death detection ELISA and a morphological study by the TUNEL reaction. As a control, LPS treatment did not induce thymocyte apoptosis in C3H/HeJ mice. Flow cytometric analysis indicated that thymus atrophy was due predominantly to the deletion of CD4+ CD8+ T cells. When cells were undergoing apoptosis, an elevation in the percentage of T-cell receptor (TCR)-alphabeta(high) cells was observed at 24 h, which was correlated with the increase in the percentages of cells expressing high levels of the Vbeta6 and Vbeta8 TCR. Gel electrophoretic analysis demonstrated the presence of protein bands with mol.wts ranging from 17 to 65 kDa in Y. enterocolitica culture supernates.

Retrograde lymphatic spread: a likely route for metastatic ovarian cancers of gastrointestinal origin.

In order to outline the pathways of gastrointestinal malignancies metastasizing to the ovaries, we reviewed 103 cases of metastatic ovarian tumors, and also performed para-aortic lymph node sampling on 11 patients at operation for metastatic ovarian tumors. Of the 103 patients, 74% (26/35) with gastric cancer and 67% (45/67) with colorectal cancer had lymph node metastasis at or before the diagnosis of ovarian tumor. Intraperitoneal metastases presented in 49 and 42% of patients with gastric and with colorectal cancers, respectively. Twenty-three percent of gastric cancer patients and 25% of colorectal cancer patients presented with both lymph node and intraperitoneal metastases. The ovary was the first or among the early metastatic organs diagnosed in 51 of the 53 patients with metachronous ovarian metastases. Only 4 patients with colorectal cancer and none with gastric cancer showed parenchymal organ metastases. These 4 patients also showed intraperitoneal lesions, and 3 of these 4 patients had node metastasis. Among the 11 patients who underwent prospective para-aortic lymph node sampling during operation for the ovarian tumors, only 1 had enlarged para-aortic nodes depicted by computed tomography, 2 had grossly enlarged (>/=1.5 cm) para-aortic lymph nodes noted at surgery, and 6 of the 7 patients with gastric cancer and all 3 with colorectal cancer had metastatic nodes histologically. Among the 58 nodes taken from these patients, 67% showed metastatic foci. We concluded that lymph node metastasis is frequently seen in patients with metastatic ovarian tumors of gastrointestinal origin, and hypothesized that retrograde lymphatic spread is a likely route for the metastases.

Phosphorylation stimulates the cooperative DNA-binding properties of the transcription factor OmpR.

The two-component regulatory proteins OmpR and EnvZ of Escherichia coli K-12 regulate expression of the major outer membrane porin protein, OmpF. OmpR is a DNA-binding protein that is involved in both the positive and negative control of ompF transcription. EnvZ is a histidine kinase that phosphorylates OmpR in response to environmental signals. We used DNA migration retardation analysis to examine the interactions of OmpR and the phosphorylated form of OmpR (OmpR-P) with the regulatory region immediately upstream of the ompF promoter. Our results indicate that the binding of OmpR to this regulatory region is cooperative and that phosphorylation significantly stimulates these cooperative interactions. Moreover, although phosphorylation increases the intrinsic binding of OmpR to a single OmpR-binding site, the primary role of phosphorylation in ompF regulation is to facilitate cooperative interactions between OmpR molecules bound at adjacent sites. Based on these results, we propose a model to explain how the phosphorylation of OmpR could stimulate the occupancy of specific sites in the ompF regulatory region, thereby resulting in the activation or repression of ompF transcription under the appropriate environmental conditions.

Identification of the bases in the ompF regulatory region, which interact with the transcription factor OmpR.

Expression of the outer membrane protein OmpF of Escherichia coli K-12 is influenced by a variety of environmental signals. Most of the signals are thought to regulate OmpF expression at the level of transcription initiation. A key element of this regulation is the interaction between the transcriptional factor OmpR and the cis-acting regulatory region of ompF. In this study, we used a combination of DNase I, dimethyl sulfate and hydroxyl radical footprinting analysis and DNA migration retardation assays to identify the bases within the ompF regulatory region that are in contact with OmpR. Our results indicate that the -107 to -39 region of ompF contains three individual binding sites and that a single OmpR-binding site is capable of interacting with two OmpR molecules. We also establish that a single OmpR-binding site is composed of two half-sites and that both half-sites are required for the formation of stable OmpR/DNA complexes. Comparisons of the sequences protected by OmpR indicate that an OmpR-binding site spans approximately 18 bp and has two highly conserved G/C base-pairs that are separated by three nucleotides. Although the three OmpR-binding sites we identified exhibit limited sequence similarity, this may reflect the fact that two of the sites are incapable of binding OmpR independently and can bind OmpR only if adjacent to another OmpR-binding site. Finally, our DNA migration retardation assays suggest that phosphorylation stimulates the cooperative interactions between OmpR molecules bound at neighboring sites. Therefore, this study provides a detailed understanding of how OmpR interacts with its binding sites immediately upstream of ompF and serves as a foundation for studying how phosphorylation of OmpR results in the regulation of ompF expression in response to environmental signals.

Sepsis-induced apoptosis of the thymocytes in mice.

Intraperitoneal injection of Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa) induces thymic atrophy in mice. The thymus weight, cell number, and viability began to decrease at 3 h, and reached their lowest level at 72 h. The thymocyte death was associated with DNA fragmentation of approximately 200 base pairs in ladder form. The kinetic study on histopathology revealed the process of thymocyte death and thymic atrophy. Flow-cytometric analysis showed that CD4+CD8+ thymocytes decreased predominantly. LPS caused thymocyte apoptosis, but only in LPS-responder mice, unlike Gram-negative bacteria that induced apoptosis in both LPS-responder (C3H/HeN) and LPS-nonresponder (C3H/HeJ). Gram-positive bacteria Streptococcus pneumoniae also caused apoptosis in LPS-nonresponder (C3H/HeJ) and LPS-responder mice (B6). The kinetics of serum TNF-alpha production after Gram-negative or Gram-positive bacteria injection was slightly different. E. coli induced serum TNF-alpha peak at 1 h in B6 mice, whereas S. pneumoniae induced a peak at 6 h in C3H/HeJ and at 9 h in B6 mice. Similarly, S. pneumoniae induced thymocyte apoptosis around 9 to 12 h, which was 6 to 9 h later than that observed with E. coli in B6 mice. Anti-TNF-alpha Ab completely blocked the E. coli-induced thymocyte apoptosis, but was only partially inhibitory on the S. pneumoniae-induced thymocyte apoptosis. Furthermore, thymocyte apoptosis induced by E. coli was inhibited by cycloheximide or actinomycin D. These data indicate that both Gram-negative and Gram-positive bacteria could induce thymus atrophy via apoptosis, and that TNF-alpha is a common denominator released and might be responsible for the thymocyte apoptosis.

A distant upstream site involved in the negative regulation of the Escherichia coli ompF gene.

The two-component regulatory system, OmpR-EnvZ, of Escherichia coli K-12 regulates the expression of the major outer membrane porin protein, OmpF. OmpR is a DNA-binding protein which acts as both an activator and a repressor to control ompF transcription. In this article, we describe a new OmpR-binding site that is located between 384 to 351 bp upstream from the ompF start point of transcription. Inactivation of this site by insertion of a 22-bp fragment prevents the repression of ompF expression conferred by the dominant negative mutation, envZ473. On the basis of the location of this binding site, the presence of bent DNA in the ompF regulatory region (T. Mizuno, Gene 54:57-64, 1987), and the fact that mutations altering integration host factor result in constitutive ompF expression (P. Tsui, V. Helu, and M. Freundlich, J. Bacteriol. 170:4950-4953, 1988), we propose that the negative regulation of ompF involves a DNA loop structure.

Effects of lysine-sensitive aspartokinase III on lysine biosynthesis in Escherichia coli K-12.

A promoterless lysC gene, coding for Escherichia coli aspartokinase III (AKase III), has been cloned by phenotypic complementation using plasmid pUC19 as the vector. The hybrid plasmid obtained, pUC19AK3, preserved the ribosome binding site and transcriptional termination signal of the gene but with a lac promoter. E. coli strains containing the recombinant plasmid had high levels of AKase III activity. AKase III activity from expressing strains was inhibited by lysine, leucine, and S-(2-aminoethyl)-L-cysteine (AEC) but not by threonine and methionine. The overexpressed AKase III enzyme had a molecular weight of about 50 kD from SDS-polyacrylamide gel electrophoresis. N-terminal amino acid sequence analysis confirmed that the product from the hybrid plasmid was identical to native AKase III rather than a fusion protein. Moreover, overexpression of AKase III significantly increased lysine excretion in the plasmid-harboring E. coli strain DH1. This increase in the level of AKase III activity also affected other metabolites than lysine. Addition of aspartate to the medium brought about significant increases in lysine excretion. A maximum increase (about 8-fold) in lysine accumulation was observed 45 minutes after incubation in minimal medium containing 0.2% aspartate as compared to aspartate-free medium.

Classification, pathogenicity, and drug susceptibility of hemolytic gram-negative bacteria isolated from sick or dead chickens.

Fifteen hemolytic gram-negative bacteria were isolated from the respiratory tracts of sick birds suffering from a long-lasting respiratory syndrome or from the bone marrow of dead birds distributed in the southern part of Taiwan. These were classified as Pseudomonas aeruginosa (10 isolates), Pseudomonas fluorescens (2 isolates), Pseudomonas stutzeri (1 isolate), Pasteurella haemolytica (1 isolate), and Proteus morganii (1 isolate). Each isolate was inoculated intraperitoneally into one group of ten 4-week-old male white leghorn chickens. Mortality and lesions were scored daily for 1 week. Three of the 10 isolates of Pseudomonas aeruginosa caused 100% mortality. Six other isolates of Pseudomonas aeruginosa and the one isolate of Proteus morganii caused 50% mortality. The remaining isolates induced less than 30% mortality. The sole nonpathogenic sample was one isolate of Pseudomonas fluorescens. When therapeutic levels of 22 antibiotics or sulfa drugs were evaluated for their inhibitory activity against the 15 isolates, the most effective were apramycin (15/15), gentamicin (15/15), spectinomycin (13/15), oxytetracycline (8/15), and sulfachloropyrazine (7/15). The least effective were ampicillin, cloxacillin, and tiamulin, which were not effective against any of the isolates. The 14 other drugs were of very low (> 4/15) effectiveness. Most of the isolates studied were virulent for chickens and very resistant to currently used drugs.

Cancer of the colon during pregnancy: report of a case and review of the literature.

A case of adenocarcinoma of the sigmoid colon during pregnancy is reported. The patient presented with anemia and a painless mass over the left abdomen without gastrointestinal discomfort, making this case different from 25 previously reported cases of colon carcinoma above the peritoneal reflection associated with pregnancy.

A study on the ecological habit of Armigeres subalbatus in Dawa area of the Mengshan Mountain in Shandong Province.

Armigeres subalbatus (A.s.) was reported in Shandong Province for the first time in 1965 and found in five counties of south Shandong including Pingyi, Linyi etc. in 1986. In Dawa area of the Mengshan mountain A.s. alults could be found in the first ten days of May, which increased in number in July, and become the dominant species in mosquito colonies in Aug. and Sept., then decreased gradually in number in Oct. and disappeared in Nov. There were two peaks of activity and blood-sucking behavior during the 24 hours of a day, one at dusk and the other at dawn. When the temperature dropped to 16 degrees C and below in the last ten days of Oct., the wigglers began their diapause period. The survival ratio reached 90.5% after 12 h freezing at -5 degrees C and none survived after 60 hours freezing. When the temperature rose to 17 degrees C and above the over-winterting larvae developed into adults, which could suck blood only at the temperature above 17.5 degrees C.