Knock-down of REL2, but not defensin A, augments Aedes aegypti susceptibility to Bacillus subtilis and Escherichia coli

Some components of the Toll and Imd immune signaling pathways have remained conserved between Drosophila and mosquitoes, however, important differences in the way invading microorganisms activate these pathways in these organisms have started to be revealed. In the present study, we have attempted to silence the Aedes aegypti NF-κB-like factor REL2, which is analogous to Drosophila Relish, and analyze the effects on mosquito mortality upon infection with a Gram-negative and a Gram-positive bacterium, both containing a DAP-type peptidoglycan, and effects on embryo development. Moreover, we have silenced one of the antimicrobial peptides (AMPs) controlled by REL2, defensin A, a major AMP in A. aegypti, and compared the results on mosquito mortality upon bacterial infection to those obtained with REL2 silencing. Results show that REL2 is crucial for A. aegypti immunity upon infection with Escherichia coli and Bacillus subtilis, corroborating with previous studies on that REL2/Imd in mosquitoes is involved in a generalized antibacterial defense, differently than its analogous in Drosophila. However, defensin A silencing did not cause a significant increase in mortality in infected mosquitoes, indicating that this peptide is not essential in mosquito protection against the two bacteria and that other immune factors controlled by REL2 are playing this role. In regard to embryo development, REL2 knock-down did not cause any significant effect on: the number of laid eggs, number of developed pupae, percent of emerged adults, and ratio between emerged females versus males. A slight decrease in the number of hatched eggs (percent eclosion) was observed in REL2 knock-down mosquitoes, but these observations were inconclusive.     

A conserved mechanism for extracellular signaling in eukaryotes and prokaryotes

Epidermal growth factor receptor (EGFr) is a key mediator of cell communication during animal development and homeostasis. In Drosophila, the signaling event is commonly regulated by the polytopic membrane protein Rhomboid (RHO), which mediates the proteolytic activation of EGFr ligands, allowing the secretion of the active signal. Until very recently, the biochemical function of RHO had remained elusive. It is now believed that Drosophila RHO is the founder member of a previously undescribed family of serine proteases, and that it could be directly responsible for the unusual, intramembranous cleavage of EGFr ligands. Here we show that the function of RHO is conserved in Gram-negative bacteria. AarA, a Providencia stuartii RHO-related protein, is active in Drosophila on the fly EGFr ligands. Vice versa, Drosophila RHO-1 can effectively rescue the bacterium's ability to produce or release the signal that activates density-dependent gene regulation (or quorum sensing). This study provides the first evidence that prokaryotic and eukaryotic RHOs could have a conserved role in cell communication and that their biochemical properties could be more similar than previously anticipated.     

Identification of bacterial microflora in the midgut of the larvae and adult of wild caught Anopheles stephensi: a step toward finding suitable paratransgenesis candidates

To describe the midgut microbial diversity and to find the candidate bacteria for the genetic manipulation for the generation of paratransgenic Anopheline mosquitoes refractory to transmission of malaria, the microbiota of wild larvae and adult Anopheles stephensi mosquito midgut from southern Iran was studied using a conventional cell-free culture technique and analysis of a 16S ribosomal RNA (rRNA) gene sequence library. Forty species in 12 genera including seven Gram-negative Myroides, Chryseobacterium, Aeromonas, Pseudomonas, Klebsiella, Enterobacter and Shewanella and five Gram-positive Exiguobacterium, Enterococcus, Kocuria, Microbacterium and Rhodococcus bacteria were identified in the microbiota of the larvae midgut. Analysis of the adult midgut microbiota revealed presence of 25 Gram-negative species in five genera including Pseudomonas, Alcaligenes, Bordetella, Myroides and Aeromonas. Pseudomonas and Exiguobacterium with a frequency of 51% and 14% at the larval stage and Pseudomonas and Aeromonas with a frequency of 54% and 20% at the adult stage were the most common midgut symbionts. Pseudomonas, Aeromonas and Myroides genera have been isolated from both larvae and adult stages indicating possible trans-stadial transmission from larva to adult stage. Fast growth in cheap media, Gram negative, and being dominantly found in both larvae and adult stages, and presence in other malaria vectors makes Pseudomonas as a proper candidate for paratransgenesis of An. stephensi and other malaria vectors.     

高通量测序技术在按蚊共生微生物研究中的应用

按蚊是疟疾的传播媒介,又称疟蚊。在按蚊体内,尤其是肠道内定殖着大量的共生微生物。共生微生物直接或间接地影响蚊虫的营养、发育、繁殖和免疫等重要生理活动。研究表明,肠道共生菌还能影响疟原虫的入侵、发育和传播。按蚊对常用杀虫剂逐渐产生抗性,疟原虫也对抗疟药产生了抗药性,严重阻碍了消除疟疾进程,迫切需要新的疟疾传播阻断战略。利用共生微生物控制和阻断疟疾传播是有潜力的方法之一,因此研究按蚊的共生微生物组成对理解按蚊、共生微生物以及病原体三者间的互作关系,识别用于防控疟疾的候选细菌有重要意义。高通量测序技术的应用有效克服了传统方法的局限性,极大地提高了对按蚊共生微生物多样性的认识。本文将从高通量测序技术在按蚊共生微生物组成、多样性和影响因素及其在疟疾防控中的应用进展进行综述。     

斯氏按蚊转录因子Relish基因克隆、定位及不同食源条件下表达

目的 对斯氏按蚊转录因子Relish基因进行克隆、定位和差异分析，初步探讨转录因子Relish在前酚氧化酶（PPO）级联与疟原虫卵囊黑化中的信号调控作用。 方法 设计简并引物，对全蚊cDNA模板进行RT-PCR扩增，产物纯化、克隆、测序和鉴定，利用特异引物分别从血细胞或中肠扩增目的基因，于不同食源条件下进行半定量分析和原位核酸分子杂交定位。 结果 获得了1种斯氏按蚊Relish cDNA部分序列，与冈比亚按蚊Relish基因很相似，并存在于中肠和血细胞，半定量分析显示，感染约氏疟原虫6、12、24和48 h或吸硝喹糖水12和24 h，于诱导约氏疟原虫卵囊黑化之前表达明显增强，原位核酸分子杂交证实血细胞和中肠有表达。 结论 斯氏按蚊转录因子Relish可能在疟原虫感染或/和约氏疟原虫卵囊黑化调控中发挥重要作用。     

应用rDNA ITS2区段基因序列分析对浙江省传疟媒介的鉴定

目的 对浙江省传疟媒介种类进行鉴定，以指导疟疾防治工作。方法 针对媒介按蚊核糖体核酸内转录间隔2（rDNAITS2）区段基因特征，应用PCR基因鉴别技术，对浙江省现场捕捉的按蚊与嗜人按蚊、八代按蚊、中华按蚊、雷氏按蚊实验室标本进行基因鉴别和比较。结果 浙江省现场捕捉的按蚊DNA样本的PCR扩增产物均为250bp，与实验室中华按蚊标本一致。结论 中华按蚊目前仍是浙江省的主要传疟媒介，现场调查未发现嗜人按蚊。     

From the Cover: Visual arrestins in olfactory pathways of Drosophila and the malaria vector mosquito Anopheles gambiae

Arrestins are important components for desensitization of G protein-coupled receptor cascades that mediate neurotransmission as well as olfactory and visual sensory reception. We have isolated AgArr1, an arrestin-encoding cDNA from the malaria vector mosquito, Anopheles gambiae, where olfaction is critical for vectorial capacity. Analysis of AgArr1 expression revealed an overlap between chemosensory and photoreceptor neurons. Furthermore, an examination of previously identified arrestins from Drosophila melanogaster exposed similar bimodal expression, and Drosophila arrestin mutants demonstrate impaired electrophysiological responses to olfactory stimuli. Thus, we show that arrestins in Drosophila are required for normal olfactory physiology in addition to their previously described role in visual signaling. These findings suggest that individual arrestins function in both olfactory and visual pathways in Dipteran insects; these genes may prove useful in the design of control strategies that target olfactory-dependent behaviors of insect disease vectors.     

Spatio-temporal distribution of malaria vectors (Diptera: Culicidae) across different climatic zones of Iran

Malaria is a main vector-borne public health problem in Iran. The last studies on Iranian mosquitoes show 31 Anopheles species including different sibling species and genotypes, eight of them are reported to play role in malaria transmission. The objective of this study is to provide a reference for malaria vectors of Iran and to map their spatial and temporal distribution in different climatic zones. Shape files of administrative boundaries and climates of Iran were provided by National Cartographic Center. Data on distribution and seasonal activity of malaria vectors were obtained from different sources and a databank in district level was created in Excel 2003, inserted to the shape files and analyzed by ArcGIS 9.2 to provide the maps. Anopheles culicifacies Giles s.l., Anopheles dthali Patton, Anopheles fluviatilis James s.l., Anopheles maculipennis Meigen s.l., Anopheles sacharovi Favre, Anopheles stephensi Liston, and Anopheles superpictus Grassi have been introduced as primary and secondary malaria vectors and Anopheles pulcherrimus Theobald as a suspected vector in Iran. Temporal distribution of anopheline mosquitoes is restricted to April-December in northern Iran, however mosquitoes can be found during the year in southern region. Spatial distribution of malaria vectors is different based on species, thus six of them (except for Anopheles maculipennis s.l. and Anopheles sacharovi) are reported from endemic malarious area in southern and southeastern areas of Iran. The climate of this part is usually warm and humid, which makes it favorable for mosquito rearing and malaria transmission. Correlation between climate conditions and vector distribution can help to predict the potential range of activity for each species and preparedness for malaria epidemics.     

Gambicin: a novel immune responsive antimicrobial peptide from the malaria vector Anopheles gambiae

A novel mosquito antimicrobial peptide, gambicin, and the corresponding gene were isolated in parallel through differential display-PCR, an expressed sequence tag (EST) project, and characterization of an antimicrobial activity in a mosquito cell line by reverse-phase chromatography. The 616-bp gambicin ORF encodes an 81-residue protein that is processed and secreted as a 61-aa mature peptide containing eight cysteines engaged in four disulfide bridges. Gambicin lacks sequence homology with other known proteins. Like other Anopheles gambiae antimicrobial peptide genes, gambicin is induced by natural or experimental infection in the midgut, fatbody, and hemocyte-like cell lines. Within the midgut, gambicin is predominantly expressed in the anterior part. Both local and systemic gambicin expression is induced during early and late stages of natural malaria infection. In vitro experiments showed that the 6.8-kDa mature peptide can kill both Gram-positive and Gram-negative bacteria, has a morphogenic effect on a filamentous fungus, and is marginally lethal to Plasmodium berghei ookinetes. An oxidized form of gambicin isolated from the cell line medium was more active against bacteria than the nonoxidized form from the same medium.     

Acute effects of dietary fat on inflammatory markers and gene expression in first-degree relatives of type 2 diabetes patients

BACKGROUND: Subjects with type 2 diabetes (T2D) and their relatives (REL) carry an increased risk of cardiovascular disease (CVD). Low-grade inflammation, an independent risk factor for CVD, is modifiable by diet. Subjects with T2D show elevated postprandial inflammatory responses to fat-rich meals, while information on postprandial inflammation in REL is sparse. AIM: To clarify whether medium-chain saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) have differential acute effects on low-grade inflammation in REL compared to controls (CON). METHODS: In randomized order, 17 REL and 17 CON ingested two fat-rich meals, with 72 energy percent from MUFA and 79 energy percent from mainly medium-chain SFA, respectively. Plasma high sensitivity C-reactive protein (hs-CRP), interleukin-6 (IL-6), adiponectin, and leptin were measured at baseline, 15 min, 60 min, and 240 min postprandially. Muscle and adipose tissue biopsies were taken at baseline and 210 min after the test meal, and expression of selected genes was analyzed. RESULTS: Plasma IL-6 increased (p < 0.001) without difference between REL and CON and between the meals, whereas plasma adiponectin and plasma hs-CRP were unchanged during the 240 min observation period. Plasma leptin decreased slightly in response to medium-chain SFA in both groups, and to MUFA in REL. Several genes were differentially regulated in muscle and adipose tissue of REL and CON. CONCLUSIONS: MUFA and medium-chain SFA elicit similar postprandial circulating inflammatory responses in REL and CON. Medium-chain SFA seems more proinflammatory than MUFA, judged by the gene expression in muscle and adipose tissue of REL and CON.     

An isoenzyme marker possibly associated with the susceptibility of Biomphalaria glabrata populations to Schistosoma mansoni

Nine laboratory populations and one field population of the snail host Biomphalaria glabrata were compared with respect to their electrophoretic patterns for acid phosphatase (AcP) and with their susceptibility to Schistosoma mansoni infection. A strong correlation (r = 0.98) was noted between the frequency of the isoenzymes AcP2-S and Acp2-F observed in the populations and the level of snail susceptibility as determined by bioassay. The isoenzyme AcP2-S was associated with susceptibility, AcP2-F with the refractory state. Breeding experiments between refractory and susceptible snails demonstrated that the refractory state was dominant and all F1 snails exhibited the AcP2-F isoenzyme and proved refractory to infection.     

2005—2019年贵州省传疟媒介按蚊密度及种群监测

目的　了解2005—2019年贵州省传疟媒介按蚊密度、种群构成及生境分布,为制定全省输入性疟疾传播风险防控措施提供参考依据。方法　2005—2019年在贵州省分别采用人帐诱法和诱蚊灯法开展按蚊密度及按蚊种群监测,对捕获的按蚊进行形态学分类鉴定并计数,并对按蚊生境分布进行分析。 结果　2005—2019年贵州省传疟按蚊密度自6月上旬开始上升,在7月上旬达到高峰后开始下降,活动规律呈单峰型,最高密度为2018年8月上旬的57.34只/（人·夜）、最低密度为2009年10月下旬的1.29只/（人·夜）;年均按蚊密度由2005年的17.91只/（人·夜）逐年缓慢降至2012年的12.34只/（人·夜）,下降了38.02%（[χ2]趋势 = 115.04,P < 0.01）,2017—2019年按蚊密度有上升趋势（[χ2] 趋势 = 420.00,P < 0.01）。2017—2019年贵州省传疟按蚊在19：00至次日7：00监测时间内均能被捕获,总体密度呈先上升后下降趋势,19：00—21：00为全省传疟按蚊活动高频时段。2005—2019年贵州省采用诱蚊灯法共监测938次,累计捕获按蚊52 781只,其中中华按蚊49 705只、微小按蚊804只、嗜人按蚊238只、其他按蚊2 034只,各类按蚊构成差异有统计学意义（[χ2] = 165.68,P < 0.01）。2017—2019年,在贵州省人房、户外和畜房三类生境中累计捕获传疟按蚊24 557只,以畜房捕获按蚊最多,占总捕获按蚊总数的67.65%;人房捕获按蚊最少,占12.01%;不同生境捕获按蚊数量差异有统计学意义（[χ2] = 55.04,P < 0.01）。在捕获的按蚊种类中,三类生境均能捕获中华按蚊、嗜人按蚊和微小按蚊,其中以中华按蚊最多,占98.07%;嗜人按蚊最少,占0.09%。结论　贵州省原疟疾流行区传疟媒介种群结构发生了改变,中华按蚊取代了微小按蚊和嗜人按蚊成为全省主要传疟媒介;传疟按蚊密度近年来有上升趋势,捕获的按蚊种类及数量均有所增加,有引起输入性疟疾本地传播的潜在风险。今后需在贵州省长期、持续、全面开展传疟媒介监测。     

Anopheles species of the mount Cameroon region: biting habits,feeding behaviour and entomological inoculation rates

There is a lack of data on the Anopheles fauna, its biology and the roles played by different vector species in the transmission of malaria in the mount Cameroon region. The biting habits, feeding behaviour and entomological inoculation rates of different Anopheles species during the dry and rainy season were investigated. A total of 2165 Anopheles was collected, 805 in the rainy season and 1360 in the dry season. Five Anopheles species were identified: Anopheles gambiae s.l., An. funestus, An. hancocki, An. moucheti and An. nili. An. gambiae, An. funestus and An. hancocki, recorded during both seasons, were the main vectors of malaria in the region. An. gambiae s.s. was the only member of the An. gambiae (Giles) complex. These three species had their peak activity between 1 and 2 am. A human blood index (HBI) of 98.29% was recorded for fed Anopheles. The sporozoite rate, for all vectors together, was significantly higher in the rainy season (9.4%) than in the dry season (4.2%) with all the species infected by Plasmodium falciparum. The average inoculation rate was 0.44 infective bites per man per night, which adds up to 161 infective bites per year in this study area. Analyses of relative abundance and infection rate of malaria vectors at different sites situated along a transect of 20 km during the dry season showed high heterogeneity in biting and sporozoite rates. No malaria vector was caught at 1200 m a.s.l. The mount Cameroon region should be considered an area of high malaria transmission intensity.     

Molecular cloning and functional expression of the first two specific insect myosuppressin receptors

The Drosophila Genome Project database contains the sequences of two genes, CG8985 and CG13803, which are predicted to code for G protein-coupled receptors. We cloned the cDNAs corresponding to these genes and found that their gene structures had not been correctly annotated. We subsequently expressed the coding regions of the two corrected receptor genes in Chinese hamster ovary cells and found that each of them coded for a receptor that could be activated by low concentrations of Drosophila myosuppressin (EC50,4 x 10(-8) M). The insect myosuppressins are decapeptides that generally inhibit insect visceral muscles. Other tested Drosophila neuropeptides did not activate the two receptors. In addition to the two Drosophila myosuppressin receptors, we identified a sequence in the genomic database from the malaria mosquito Anopheles gambiae that also very likely codes for a myosuppressin receptor. To our knowledge, this paper is the first report on the molecular identification of specific insect myosuppressin receptors.