Introduction: Bacillus cereus (B. cereus) is a common gram-positive bacterium that contaminates starch-rich food and can cause outbreaks of foodborne diseases. This study describes the characteristics of outbreaks caused by B. cereus in China during 2010-2020 and explore the possible reasons for changes in the number of outbreaks over time. Results of this analysis can efficiently help guide and allocate public resources to prevent B. cereus-caused foodborne diseases.Methods: Descriptive statistical methods were used to analyze the data on B. cereus outbreaks in China during this period. The data were identified and reported at all levels in China through National Foodborne Disease Outbreak Surveillance System.Results: From 2010 to 2020, a total of 419 foodborne outbreaks prompted by B. cereus were reported in China, leading to 7,892 cases, 2,786 hospital admissions, and 5 fatalities. The bulk of the outbreaks were recorded in the summer, primarily between May and September. The most recurrent food vehicle was linked with rice or flour-based products, notably those made with rice or fried rice. School canteens bore the brunt of the B. cereus outbreaks. In multifactor outbreaks, food contamination was identified as the most common culprit; while in instances where only one factor contributed, improper storage was most frequently implicated.Conclusion: The prevalence of B. cereus outbreaks remained relatively consistent throughout the studied period. Understanding the types of foods, causative factors, and contributing elements leading to B. cereus outbreaks can help inform prevention strategies for foodborne illnesses. The majority of outbreaks were associated with rice-or flour-based foods in school canteens, suggesting contamination and improper storage during food preparation. Consequently, it is essential to prioritize continuous education for canteen staff on food safety, efficacious management, and proper practices. The implementation of comprehensive guidelines, encompassing multiple critical aspects, can potentially reduce the occurrence of B. cereus outbreaks.

Invasive listeriosis is a rare but serious foodborne disease that causes maternal-neonatal, central nervous system, and bloodstream infections. The aim of this study was to assess the whole-genome sequencing (WGS)-based genetic diversity of clinical Listeria monocytogenes isolates over a 7-year period and prove the effect of WGS application in food vehicle investigation. A total of 360 isolates were recovered during 2013 and 2019 through the national listeriosis special surveillance program. Two hundred twenty-six isolates (62.8%) were associated with pregnancy. All isolates belonged to lineage I (214 isolates) or lineage II (146 isolates), with 4 serogroups (46.9% IIb, 39.7% IIa, 12.5% IVb, and 0.8% IIc). All isolates were in 25 clonal complexes (CCs) and 3 singletons, with CC87, CC8, and CC5 being the most common causes of human listeriosis. All clinical isolates were positive for Listeria pathogenicity island 1 (LIPI-1), LIPI-3 was present in 21.4% of isolates and LIPI-4 was detected in 29.2% of isolates. LIPI-4-positive isolates, including CC87, sequence type (ST)619, ST382, CC4, and CC2, have been shown to confer hypervirulence. Fifteen isolates harbored at least one antimicrobial encoding gene, including tet (M), mef (A), msr (D), and dfr (G). The sublineage designations were consistent with CC designations, and 215 distinct cgMLST types (CTs) were classified, the most abundant being CT58 and CT750. In summary, there is a high level of genetic diversity among the clinical isolates. WGS has strengthened listeriosis surveillance and will be implemented for other foodborne bacteria in the National Molecular Tracing Network for Foodborne Disease.

BACKGROUND:The combination of Wolbachia-based incompatible insect technique (IIT) and radiation-based sterile insect technique (SIT) can be used for population suppression of Aedes aegypti. Our main objective was to evaluate whether open-field mass-releases of wAlbB-infected Ae. aegypti males, as part of an Integrated Vector Management (IVM) plan led by the Mexican Ministry of Health, could suppress natural populations of Ae. aegypti in urbanized settings in south Mexico.METHODOLOGY/PRINCIPAL FINDINGS:We implemented a controlled before-and-after quasi-experimental study in two suburban localities of Yucatan (Mexico): San Pedro Chimay (SPC), which received IIT-SIT, and San Antonio Tahdzibichén used as control. Release of wAlbB Ae. aegypti males at SPC extended for 6 months (July-December 2019), covering the period of higher Ae. aegypti abundance. Entomological indicators included egg hatching rates and outdoor/indoor adult females collected at the release and control sites. Approximately 1,270,000 lab-produced wAlbB-infected Ae. aegypti males were released in the 50-ha treatment area (2,000 wAlbB Ae. aegypti males per hectare twice a week in two different release days, totaling 200,000 male mosquitoes per week). The efficacy of IIT-SIT in suppressing indoor female Ae. aegypti density (quantified from a generalized linear mixed model showing a statistically significant reduction in treatment versus control areas) was 90.9% a month after initiation of the suppression phase, 47.7% two months after (when number of released males was reduced in 50% to match local abundance), 61.4% four months after (when initial number of released males was re-established), 88.4% five months after and 89.4% at six months after the initiation of the suppression phase. A proportional, but lower, reduction in outdoor female Ae. aegypti was also quantified (range, 50.0-75.2% suppression).CONCLUSIONS/SIGNIFICANCE:Our study, the first open-field pilot implementation of Wolbachia IIT-SIT in Mexico and Latin-America, confirms that inundative male releases can significantly reduce natural populations of Ae. aegypti. More importantly, we present successful pilot results of the integration of Wolbachia IIT-SIT within a IVM plan implemented by Ministry of Health personnel.

Introduction: Mushroom poisoning was the leading cause of foodborne disease outbreaks and outbreak-associated deaths in China. Mushroom poisoning outbreak surveillance can provide insight into the epidemiological characteristics of mushroom poisonings and guide policymaking and health education to reduce illnesses and deaths. Methods: Foodborne Disease Outbreak Surveillance System was upgraded in 2011 to collect foodborne disease outbreaks in China. Mushroom poisoning outbreaks during 2010-2020 were selected to analyze geographical distribution, seasonal distribution, and setting of food preparation. Results: A total of 10,036 outbreaks, which resulted in 38,676 illnesses and 788 deaths, were reported in this period. Mushroom poisonings occurred all over the country, but with highest incidence in the southwest and central China. Overall, 84.6% outbreaks were associated with food prepared in households, followed by 8.7% in street stalls, and 2.5% in canteens. Mushroom poisoning outbreaks clearly exhibited seasonality, and the peak season was summer through autumn. Outbreaks occurring between May and October accounted for 94.1% of total outbreaks, 92.4% illnesses, and 97.2% deaths. Conclusions: Mushroom poisoning is a food safety issue of higher concern in China. Targeted health education is essential to reduce mushroom poisoning, especially in southwest China. Citizens are advised to not collect or eat wild mushrooms.

What is already known on this topic? Vibrio parahaemolyticus (V. parahaemolyticus) is frequently resistant to common antimicrobials such as ampicillin and generally highly susceptible to most clinically used antimicrobials. What is added by this report? V. parahaemolyticus were highly resistant to cefazolin and ampicillin: 94.4% and 37.0%, respectively. However, it was below 3% resistance to all 10 other antimicrobials including clinically relevant agents and even imipenem. The overall levels of antimicrobial resistance and multidrug resistance were 95.1% and 3.3%, respectively. The distribution of antimicrobial resistance and the multidrug resistance had regional, temporal, sexual, and isolated source strain variation. What are the implications for public health practice? This study provides data on drug resistance of V. parahaemolyticus in Chinese clinical settings, which will help develop a public health strategy.

目的 应用营养筛查量表(NSI)调查养老机构老年人营养不良风险并分析其影响因素.方法 采用方便抽样对5个城市6家养老机构中入住的410名老年人进行问卷调查.结果 养老机构老年人营养不良风险平均得分(3.43±2.45)分;营养状况良好168名(41.0％),中度营养不良风险164名(40.0％),高度营养不良风险78名(19.0％);医疗保险类型、咀嚼能力、处方药种数、患病种数、膳食类型、年龄、文化程度、牙齿情况、小腿围、握力、性别是养老机构老年人营养不良风险的影响因素.结论 养老机构老年人营养不良风险比例较高,加强对养老机构老年人营养筛查与评估,提供营养均衡、易于进食的膳食、宣传营养与健康知识、加强慢病管理及合理使用药物将有利于降低养老机构老年人营养不良风险.

Background: Candida albicans is associated with high mortality among immunocompromised patients. Resistance to and toxic side effects of antifungal drugs require the development of alternative antifungal agents. AMP-17 is a novel antimicrobial peptide derived from Musca domestica that exerts excellent antifungal effects against the Candida species. In this article, we discuss the potential mechanism of AMP-17 against C. albicans from the perspective of affecting the latter's cell external structure. Methods: Recombinant AMP-17 was prepared by prokaryotic expression system, and its anti-C. albicans activity was detected by microdilution method. Microscopy and scanning electron microscopy were used to examine morphological changes in C. albicans. Cell wall-specific staining method was used to detect the change of cell wall integrity of C. albicans after AMP-17 treatment. AMP-17-induced damage to the C. albicans cell membrane was analyzed by fluorescent probes and glycerol assay kit. The expression of genes related to fungal cell wall and cell-membrane synthesis was detected by qRT-PCR. Results: Morphological observations showed that the growth of C. albicans was significantly inhibited in AMP-17-treated cells; the cells appeared aggregated and dissolved, with severe irregularities in shape. Furthermore, AMP-17 damaged the integrity of C. albicans cell walls. The cell wall integrity rate of AMP-17-treated cells was only 21.7% compared to untreated cells. Moreover, the change of membrane dynamics and permeability suggested that the cell membrane was disrupted by AMP-17 treatment. Genetic analysis showed that after AMP-17 treatment, the cell wall synthesis-related gene FKS2 of C. albicans was up-regulated 3.46-fold, while the cell membrane ergosterol synthesis-related genes ERG1, ERG5, ERG6, and MET6 were down-regulated 5.88-, 17.54-, 13.33-, and 7.14-fold, respectively. Conclusion: AMP-17 treatment disrupted the cell wall integrity and membrane structure of C. albicans and is likely a novel therapeutic option for prevention and control of C. albicans infections.

As traditional approaches to the control of dengue and Zika are insufficient, significant efforts have been made to develop utilization of the endosymbiotic bacterium Wolbachia to reduce the ability of mosquitoes to transmit pathogens. Although Wolbachia is known to inhibit flaviviruses in mosquitoes, including dengue virus (DENV) and Zika virus (ZIKV), it remains unclear how the endosymbiont interferes with viral replication cycle. In this study, we have carried out viral binding assays to investigate the impact of the Wolbachia strain wAlbB on the attachment of DENV serotype 2 (DENV-2) and ZIKV to Aedes aegypti Aag-2 cells. RNA interference (RNAi) was used to silence a variety of putative mosquito receptors of DENV that were differentially regulated by wAlbB in Aag-2 cells, in order to identify host factors involved in the inhibition of viral binding. Our results showed that, in addition to suppression of viral replication, Wolbachia strongly inhibited binding of both DENV-2 and ZIKV to Aag-2 cells. Moreover, the expression of two putative mosquito DENV receptors – dystroglycan and tubulin – was downregulated by wAlbB, and their knock-down resulted in the inhibition of DENV-2 binding to Aag-2 cells. These results will aid in understanding the Wolbachia-DENV interactions in mosquito and the development of novel control strategies for mosquito-borne diseases.

[背景]AMP-17是从微生物诱导的家蝇转录组数据库筛选到的一条特异性高表达基因,采用原核表达体系获得其重组蛋白并证实了具有显著的抗菌效果,特别是对白色念珠菌具有较强的抗菌活性.[目的]研究抗菌肽AMP-17对白色念珠菌菌丝的抑制作用.[方法]采用微量液体稀释法测定AMP-17对11株白色念珠菌的最小抑菌浓度(minimal inhibitory concentration,MIC);根据对AMP-17的敏感程度选取3株绘制生长曲线;通过光学显微镜观察并计数经AMP-17作用后白色念珠菌芽生孢子生成率及芽管形成率;倒置荧光显微镜观察白色念珠菌酵母相向菌丝相转化及以菌丝相为起点AMP-17促进菌丝相转化为酵母相的情况.[结果]AMP-17对支气管肺泡灌洗液分离株16105的MIC为10 μg/mL,对粪便分离株16214的MIC为40 μg/mL,对其余9株白色念珠菌的MIC均为20 μg/mL;白色念珠菌经不同浓度的AMP-17作用后,各时间点的芽生孢子生成率均显著低于对照组,尤其是40 μg/mL的AMP-17组,芽生孢子生成率仅15％,显著低于阳性药物氟康唑;各实验组芽管形成率显著低于对照组,且芽管形成缓慢,培养6h后芽管形成率仅为6％,形成的芽管长度较短,仅为菌体的1-2倍.镜下观察低浓度的AMP-17即可以完全抑制白色念珠菌菌丝的生长,并且对已经形成的菌丝有一定的生长抑制作用,高浓度的AMP-17则可使已形成的部分菌丝向酵母相转化.[结论]家蝇抗菌肽AMP-17可抑制白色念珠菌菌丝生长.

Abstract Antimicrobial peptides (AMPs) are cationic small peptide chains that have good antimicrobial activity against a variety of bacteria, fungi, and viruses. AMP-17 is a recombinant insect AMP obtained by a prokaryotic expression system. However, the full antifungal activity, physicochemical characteristics, and cytotoxicity of AMP-17 were previously unknown. AMP-17 was shown to have good antifungal activity against five pathogenic fungi, with minimum inhibitory concentrations (MIC) of 9.375–18.75 μg/ml, and minimum fungicidal concentrations (MFC) of 18.75–37.5 μg/ml. Notably, the antifungal activity of AMP-17 against Cryptococcus neoformans was superior to that of other Candida spp. In addition, the hemolytic rate of AMP-17 was only 1.47%, even at the high concentration of 16× MIC. AMP-17 was insensitive to temperature and high salt ion concentration, with temperatures of 98°C and –80°C, and NaCl and MgCl2 concentrations of 50–200 mmol/l, having no significant effect on antifungal activity. However, AMP-17 was sensitive to proteases, trypsin, pepsin, and proteinase K. The elucidation of antifungal activity, physicochemical properties and cytotoxicity of AMP-17 provided an experimental basis for its safety evaluation and application, as well as indicated that AMP-17 might be a promising drug.

OBJECTIVE:To describe the hypertension awareness, treatment and control among adults in China in 2010-2012.METHODS:Data was from the China Nutrition and Health Surveillance in 2010-2012. A stratified multistage cluster sampling was used from 31 provinces, autonomous regions, and municipalities and selected 150 districts/counties. The sample was adults aged 18 and over selected through the method of Probability Proportion to Size(PPS). The mercury sphygmomanometer was used to measure the blood pressure. Each person had three measurements. The questionnaire was used to collect the information of hypertension awareness and control.RESULTS:The sample participants were 120 428 in 2010-2012. Among adults with hypertension, 46. 5%were aware of their hypertension, 41. 1% reported taking hypertension medicine in the last two weeks, and 13. 8% controlled the blood pressure. It was higher for hypertension awareness, treatment and control in women(49. 5%, 44. 2% and 14. 6%) than those of men(43. 0%, 37. 4% and 12. 9%). The three rates were increased with age. The prevalence of control was 33. 6% among the treated hypertensive individuals. It was higher for men and the rate was decreased with age. It was higher for hypertension awareness, treatment and control among hypertension and control among treated hypertensive participates in urban area(52. 7%, 47. 9%, 17. 9% and 37. 3%) than those in rural area(39. 5%, 33. 4%, 9. 2% and 27. 6%). The rates of overall, gender, and age groups were decreased in large city, medium and small city, general county, and poor county.CONCLUSION:The prevalence of hypertension awareness, treatment and control among hypertension and control among treated hypertensive participates among Chinese adults was increased in 2010-2012. But the rates were still keep a low level. The region and age differences need pay attention.

目的 从家蝇的基因组库中筛选几丁质酶2 (MDCht2)基因,进行cDNA克隆及分子特性分析,对其时空表达模式进行初步探索. 方法 从家蝇基因组数据库中筛选MDCht2基因,以该基因的cDNA为模板进行PCR扩增,采用生物信息学相关软件对MDCht2基因及其编码蛋白质的基本理化性质、信号肽、蛋白质结构等进行分析,预测蛋白质功能.取家蝇不同生活史时期(卵,1龄幼虫,2龄幼虫,3龄幼虫,蛹,雌雄成虫)及3龄幼虫不同组织(体壁,气管,马氏管,唾液腺,脂肪体,肠道)标本,采用实时荧光定量PCR检测MDCht2基因表达情况. 结果 MDCht2基因cDNA全长1 932 bp,ORF框全长1 530 bp,编码509个氨基酸,理论相对分子质量为57.8×103,pI 5.67,属于亲水性的酸性蛋白,有信号肽,功能结构域位于第37～385位氨基酸间,无几丁质结合域.二级结构分析显示存在无规则卷曲(Cc),α螺旋(Hh),β折叠(Ee)3种类型.PCR扩增得到MDCht2基因长为1 530 bp的序列片段.实时荧光定量PCR检测家蝇MDCht2基因在蛹期的表达量较卵期上调6.477 01倍(P＜0.01),3龄幼虫表达量上调2.655 25倍(P＜0.01),1龄幼虫的表达量上调2.475 01倍(P＜0.05),表达水平排列顺序为蛹＞3龄幼虫＞1龄幼虫＞2龄幼虫＞卵＞雄成虫＞雌成虫.以体壁为参照,MDCht2基因家蝇气管中的表达量较高,气管中的表达量较体壁1.81.816 51倍(P＜0.01),表达水平为气管＞体壁＞唾液腺＞脂肪体＞肠道＞马氏管. 结论 成功克隆了家蝇的MDCht2基因并初步探索了其时空表达模式,即MDCht2基因在蛹期及气管组织高表达,为MDCht2的功能研究奠定了基础.

To improve the early detection of geographically dispersed common-source foodborne outbreaks, a national foodborne disease molecular tracing network (TraNet) was launched in 2013 based on the existing laboratory-based foodborne disease surveillance system in China. TraNet consists of 32 provincial and many municipal Centers for Disease Control and Prevention that conduct molecular subtyping using pulsed-field gel electrophoresis (PFGE) on foodborne pathogens submitted by sentinel hospitals within their jurisdictions, and submit these PFGE profiles to a national database for further analysis. Currently, TraNet plays a significant role in the identification of etiologic causes and tracks contaminated food during foodborne outbreaks.

Objective To describe the age-standardized prevalence of hypertension based on the value from mercury sphygmomanometer and converted electronic sphygmomanometer among adults 18 years old and over in China 2010-2012.Methods Data was from the Chinese Nutrition and Health Surveillance in 2010-2012.The total number was 120 428 which selected through the method of multi-stage stratified cluster randomization sampling and probability proportion to size (PPS).The study objects were adults aged 18 and over in 31 Provinces and 150 sites in China Mainland.There were four categories areas:large cities,medium and small cities,general rural areas and poor rural areas.An average of blood pressure value was calculated though three systolic and diastolic blood pressure readings taken by mercury sphygmomanometer.The value of converted electronic sphygmomanometer was from a survey.Hypertension was defined as systolic blood pressure ≥ 140 mmHg and/or diastolic blood pressure ≥90 mmHg and/or use of antihypertensive drug in the past two weeks.Age-standardized result were calculated incorporating a complex sample weighting using the population data from the National Bureau of Statistics in 2009.Results The overall adjusted prevalence of hypertension based on the value of mercury sphygmomanometer among Chinese adults aged 18 and over was 22.8％(95％CI21.3％-24.3％)in 2011-2012.It was 24.1％ (95％ CI 22.3％-26.0％)in men and 21.4％ (95％ CI 20.0％-22.9％) in women.It showed 9.7％ (95％ CI 8.6％-10.9％),32.7％ (95％ CI 30.8％-34.5％) and 52.6％ (95％ CI 51.0％-54.3％) in 18-44,45-59 and 60 + age groups.Hypertension was 24.5％ (95％ CI 22.4％-26.7％) in urban and 21.1％ (95％ CI 19.0％-23.2％)in rural.The prevalence of hypertension based on the value of converted electronic device was 25.2％ (95％ CI23.6％-26.7％).It was 26.2％ (95％ CI24.4％-27.9％)in men and 24.1％(95％CI22.6％-25.6％)in women.10.6％ (95％CI9.4％-11.8％),35.7％ (95％CI 34.0％-37.5％)and 58.9％ (95％CI 57.0％-60.7％)was found in 18-44,45-59 and 60 + age groups.Prevalent hypertension was 26.8％ (95％ CI 24.7％-28.8％) in urban and 23.5％ (95％ CI 21.2％-25.8％)in rural area.Conclusion Prevalence of hypertension among the adults in China is increasing.The prevalence of hypertension from mercury sphygmomanometer is lower than that of converted electronic value.Both kinds of value have similar characteristics.More men than women have a hypertension.There is a higher prevalence as growth of age.There is a decreasing prevalence of hypertension in large cities,medium and small cities,general rural areas and poor rural areas.

Listeria monocytogenes, a ubiquitous bacterium in nature, can lead to human listeriosis through food consumption. Listeriosis is a rare, preventable, and treatable foodborne disease but can cause hospitalizations and fatalities. We reviewed the literature published in China to better understand the prevalence of L. monocytogenes in food products, incidence of human listeriosis, and characteristics of L. monocytogenes strains in China. The average prevalence of L. monocytogenes in Chinese food products in 28 provinces was 4.42%, with the highest prevalence of 8.91% in meat-poultry products, followed by aquatic animals, Chinese salad and salad, rice and flour products, and so on. Two hundred fifty-three invasive listeriosis cases were reported from 2011 to 2016 in 19 provinces, and the overall case-fatality rate was 25.7% with no deaths reported of pregnant women and children. L. monocytogenes strains were generally susceptible to most antibiotics, with ampicillin and penicillin G still effective in treatment. The predominant sequence types (STs) in food were ST9 and ST8, while clinically ST87 was most common ST in China. The national human listeriosis pilot surveillance started in 2013, and a total of 133 listeriosis cases have been collected until now. On the basis of the surveillance program, further research should be conducted to uncover the reason for the prevalence and pathogenic mechanism of the highly epidemiological hypervirulent ST87 strains in China.

克隆家蝇内源性β-葡萄糖苷酶(beta-glucosidase,BG)基因,建立两种原核表达体系和一种真核表达体系,分别检测表达产物的活性,比较不同表达体系对其表达水平及重组蛋白酶学性质的影响,为进一步研究和利用家蝇BG酶奠定基础.本研究根据BG酶基因的cDNA序列设计引物扩增BG酶基因成熟肽的基因片段,分别采用表达载体pET-28a(+)和pEGX-4T-1构建原核表达体系并在大肠杆菌BL21 (DE3)中诱导表达.在65 kDa附近均出现特异性蛋白条带,进行Western Blotting鉴定证实重组质粒在其宿主菌E.coli BL21(DE3)中成功表达,命名为MDBG-1蛋白和MDBG-2蛋白.同时选用真核表达载体pPICZaA构建酵母分泌型表达体系在酵母细胞GS115中获得稳定的表达,将其命名为MDBG-3蛋白.采用七叶苷平板法和DNS法对三种表达体系所重组表达的蛋白进行酶活性鉴定和检测,显示3种表达体系所表达的融合蛋白均具有BG酶活性,其酶活力有所不同,且真核表达体系所表达的蛋白酶活性最高.本研究对家蝇β-葡萄糖苷酶基因表达的重组蛋白特性进行初步研究,为发现新的有效纤维素酶体系提供基础数据.

Antimicrobial peptides/proteins are immune-related molecules that are widely distributed in bacteria, fungi, plants, invertebrates and higher animals. They have exhibited great potential to be developed into antimicrobial drugs. The housefly, Musca domestica, lives in a highly contaminated environment and has adapted a robust immune system against various pathogens. As an effort to search for new antimicrobial molecules in the housefly, we investigated the function of an uncharacterized gene firstly by confirming that its expression was induced by infection in M. domestica. The corresponding protein was then shown to have potent antimicrobial activity. Scanning Electron Microscopy data showed that treatment of C. albicans cells with the protein caused cell size decreasing and cell elongation. The results here suggest the protein a novel class of antimicrobial protein and provide new insights into the immunological mechanisms by which M. domestica combats invading C. albicans.

The aims of this study are to isolate the chitinase gene MDCH from Muscadomestica EST sequencing database,to clone the gene and analyze its molecular characteristic,and to explore the temporal-spatial expression patterns of the gene in different tissues and different developmental stages as well as after induced by different microorganism.Firstly,EST sequencing technology was employed to screen the chitinase gene MDCH from the constructed cDNA plasmid library of M.domestica larvae,the bioinformatics method to analyze the gene sequence and physicochemical properties of the encoded protein,and the neighbor joining to build phylogenetic tree.Then,the target gene amplified by PCRthen was constructed into the recombinant plasmid pEASY-E1-MDCⅡ,the recombinant plasmid was transformed in clonal cell Trans1-T1,and the real-time PCR technology was to detect the expression difference of the MDCⅡ gene in different developmental stages and different tissues Finaly,injection method was used to irduce different microorganisms into the 3 instar larvae ofM.domestica,and realtime PCR to detect the changes of expression level in different time points after inducing.The results indicated that the ORF length of the MDCⅡ gene was 1 374 bp,encoding 457 amino acids,and the molecular weight 51.6 kD.The alignment analysis of the phylogenetic tree revealed that the genetic distance of MDCⅡ was close to the adult growth factor of Drosophila.The recombinant plasmid pEASY-E1-MDCⅡ with correct gene sequence was successfully constructed.The MDCⅡ gene expressed in different developmental stages of M.domestica at different exprssion levels,and the highest in salivary glands and fat body of 3 instar larva.The MDCⅡgenepresented up-regulated expression at 3 hafter induction of Candida albicans,Staphylococcus aureus,and Escherichia coli.In conclusion,the MDCⅡ gene is the adult growth factor in chitinase,participating in growth and development ofM.domestica,and also playing a certain role in immune defense process.

Objective To bioinformatically analyze,clone,and determine the patterns of expression of the AMP17 gene of Musca domestica.Methods A transcriptome database was searched for M.domestica highly expressing the AMP17 gene.The gene and the protein it codes for were analyzed bioinformatically.Specimens of M.domestica were collected during different developmental stages (eggs,three instar larvae,pupae,adult females,and adult males).Specimens were collected from different sites in three instar larvae (the integument,the trachea,salivary glands,the fat body,Malpighian tubules,and the midgut).The patterns of AMP17 gene expression were detected with real-time quantitative PCR (real-time PCR).Results A specific AMP17 gene fragment about 495 bp in length was obtained after amplification with PCR.Bioinformatic analysis indicated that full-length ORF of MSP was 495 bp,and the AMP17 gene encoded 164 amino acids.AMP17 has a molecular weight of 17.4× 103 and a pI of 6.09.The polypeptide is hydrophilic and it is a secretory protein found mainly outside cells (but possibly in cell walls).Protein kinase phosphorylation sites were identified as 5 serines,3 threonines,and 1 tryptophan.The secondary structure of MSP consisted of alpha-helices,random coils,and extended strands.Spatial and temporal patterns of AMP17 expression indicated that expression of the AMP17 gene differed in each stage M.domestica development.With eggs as a reference,the level of AMP17 expression was higher in third instar larvae and males.The level of expression was highest in third instar larvae,followed by adult males,then first instar larvae,adult females,pupa,second instar larvae,and finally eggs.The level of AMP17 expression in third instar larvae was 20 320.98 times that in eggs (P＜0.01);the level of AMP17 expression in adult males was 10 936.37 times (P＜0.01) that in eggs.Levels of AMP17 expression in Malpighian tubules and salivary glands were significantly higher than those in other tissues.The level of AMP17 expression in Malpighian tubules was 2.40 times (P ＜0.05) that in the integument;the level of AMP17 expression in salivary glands was 1.31 times (P＜0.01) that in the integument.Spatial and temporal patterns of AMP17 expression indicated that expression of the AMP17 gene differed in each stage of M.domestica development.The level of AMP17 expression was higher in third instar larvae and adult males.The level of AMP17 expression in third instar larvae was significantly higher in Malpighian tubules and salivary glands than that in other tissues.Conclusion This study successfully cloned the AMP17 gene and it preliminarily examined patterns of that gene's expression.These findings provide a basis for further study of the function of AMP17.

Endogenous β-glycosidase (BG) gene of Musca domestica was cloned to construct a prokaryotic expression system, and the activities of expression products were detected. The characteristics of endogenous BG were explored to provide molecular and biological bases for further explaining the strong adaptive capacity of M.domestica to the environment and taking new measures for population control. Using the BG gene sequences of 6 representative, well-studied insects such as Spodoptera frugiperda as references, the whole-length cDNA sequence of BG gene that was cloned from cDNA of M.domestica was subjected to bioinformatics analysis by homology-based cloning in combination with RACE-PCR. Afterwards, pET-28(+) vector was used to construct a prokaryotic expression system that was transformed into E.coli BL21/DE to induce expressions of recombinant proteins. The BG activities of different recombinant proteins were detected by the aesculin plate method and DNS method, and their characteristics were preliminarily analyzed. Whole-length cDNA sequence (1933 bp) of BG gene was cloned from M.domestica, and deduced to be a polypeptide comprising 562 amino acid residues. After induced expression of recombinant prokaryotic plasmid, a specific band appeared at about 65 kDa, verifying that the recombinant plasmid was successfully expressed. The recombinant BG of M.domestica simultaneously had the activities of endo-β-1,4 glucanase (EC 3.2.1.4), exo-β-1,4-glucanase (EC 3.3.1.91) and BG (EC 3.2.1.21). BG from M.domestica is a novel multifunctional cellulase, as an important supplement for studies on inst cellulases.