Heart mechanical and hemodynamic parameters of a beetle,Tenebrio molitor,at selected ages

The physiological processes that occur during the aging of insects are poorly understood. The aim of this study was to describe the changes in contractile activity and hemodynamic parameters of the heart that take place as the coleopteran beetle, Tenebrio molitor, ages. The frequency of heart contractions in beetles that had just undergone metamorphosis (median 24.7 beats/min) was significantly lower than the frequency of heart contractions in older beetles. In 56% of beetles that were < 1 week of age, a pattern of contractile activity with alternating periods of higher and lower contraction frequency was detected, suggesting that some posteclosion developmental processes occur during the first week of adulthood. All beetles that were 1 week of age showed a regular rhythm of heart contractions (median 72 beats/min). In older beetles, abnormalities such as heart arrhythmias or heart arrest were observed. The incidence of arrhythmia as well as the arrhythmicity index was highest in beetles that were 8–18 weeks old. The calculated stroke volume (SV) was also found to increase from eclosion to 12 weeks of age, and then decreased as adults aged further. Interestingly, cardiac output increased gradually, but the ejection fraction did not change significantly with age.     

黄粉虫防御性分泌物抑菌活性的研究

目的:研究黄粉虫防御性分泌物的化学成分及其抑菌活性。方法:用二氯甲烷萃取分泌物并经GC/MS分析,后用牛津杯法和平板连续稀释法分别就该分泌物对大肠杆菌、金黄色葡萄球菌、白色假丝酵母、黑曲霉、桔青霉的抑菌作用及2-甲基对苯醌和对甲酚标准品对上述供试菌的最低抑菌浓度进行测定和比较。结果:分泌物含2-甲基对苯醌、对甲酚和正二十三烷等7种成分,抑菌强度:桔青霉>大肠杆菌和金黄色葡萄球菌>白色假丝酵母>黑曲霉。结论:该分泌物对这5种供试菌的抑制效果优于2-甲基对苯醌和对甲酚标准品。     

Periodic muscular activity and its possible functions in pupae of Tenebrio molitor

Abstract. During pupal development, Tenebrio molitor L. show regular periods of rhythmic muscular contractions and associated body movements. These periods of activity last 2.5-5.8 min and are more frequent in newly ecdysed pupae ( c. 3h^-1). They become less frequent ( c. 1.5 h^-1) when the basal metabolism reaches its lowest level. In the pharate adult stage the clear pattern of muscular activity disappears.
Muscular activity is temperature-dependent and is commonly absent below 20C. Muscular activity did not disturb the cyclic output of CO₂, which is characteristic of metamorphosis. The heart shows characteristic periods (1–3 min) of activity during pupal development. The frequency of these heart pulsation periods depends on metabolic rate. Heart pumping was correlated mostly with muscular contractions. Therefore we suggest that the main physiological function of muscular activity is to support circulation.     

黄粉甲幼虫抗菌物质的诱导及其抗菌活性

采用饥饿法、紫外线照射法和针刺法处理黄粉甲Tenebriomolitor 6龄幼虫后均能诱导其 产生抗菌物质,收集的血淋巴上清液对真菌有抑制作用,对细菌无抑制作用;经热处理后的血 淋巴上清液则对细菌有抑制作用,而对真菌无抑制作用。SDS-PAGE检测结果发现,与未诱导的 对照相比经诱导的黄粉甲幼虫血淋巴中,原有的一类大分子蛋白质如分子量分别为97kD、44 kD和37 kD左右的蛋白质缺失;而ESI-MS分析结果显示诱导后比诱导前黄粉甲幼虫血淋巴中有 小分子物质产生,推测可能是此类缺失蛋白质分解为小分子量的抗菌肽,从而表现出抗菌活性 。     

黄粉虫抗茵肽TmAMP3在大肠杆菌中的高效表达及活性检测

昆虫抗菌肽具有广谱抗菌活性,克隆黄粉虫Tenebrio molitor抗菌肽基因,进行原核表达和活性检测,为昆虫抗菌肽推广应用奠定一定基础.根据GenBank公布的黄粉虫抗菌肽序列设计特异引物,以RT-PCR法从黄粉虫体内克隆了抗菌肽基因TmAMP3,将其亚克隆至pET-30a表达载体中,转化到大肠杆菌Escheric...     

黄粉虫幼虫营养成分分析和保健功能的实验研究

对黄粉虫幼虫干粉的营养成分分析显示,其蛋白质和氨基酸含量分别占干重的５９．７０％和５９．５９％;不饱和脂肪酸占总脂肪酸含量的７７．０５％,其中仅亚油酸就占总脂肪酸含量的４１．７０％。有饰物 含量均低于国家标准。有机硒含量高达３４μｇ／１００ｇ。维生素Ａ和Ｅ含量分别为３３７和８９８μｇ／１００ｇ。实验表明,干粉液无毒性。具有较好的抗疲劳,延缓衰老和降低血清胆固醇的功能,并能提高小鼠外周淋巴细胞转化率     

黄粉虫防卫物质苯醌的测定及其去除

采用分光光度法测定了黄粉虫的防卫毒素苯醌的含量。结果表明 :各虫态各性别的黄粉虫在不同发育阶段其苯醌含量均有不同 ,各虫态的苯醌含量随日龄而增大 ,雌蛹和雌成虫的苯醌含量均高于同一日龄的雄蛹和雄成虫。经刺激后各虫态的苯醌含量升高 2 0 72 %～ 41 64%,刺激后用水清洗的虫体其体内苯醌含量下降 88 2 7%～ 92 60 %;烘烤也可使苯醌大量挥发和分解 ,采用刺激 -水洗 -研磨 -烘烤工艺处理幼虫可清除 94 3 0 %苯醌 ,此法可为保障黄粉虫作为饲用昆虫的安全性提供依据     

黄粉虫成虫繁殖力及影响幼虫发育的因素

对单配或一雄多雌或经历不同交配历期的雌黄粉虫 Ｔｅｎｅｂｒｉｏ ｍｏｌｉｔｏｒ Ｌ．产卵量进行了研究,以期了解两性的繁殖力,为雌虫多产高质量卵提供科学依据。在幼虫期添加少量鲜马铃薯可极大地促进其生长。适当的高密度和不筛粪有利于低龄幼虫生长,４０日龄以后应定期筛粪并添加新饲料。幼虫的最适密度取决于其拥有的饲料量。幼虫生态生长效率高达３１．８％。     

黄粉甲抗冻蛋白AFP84a在大肠杆菌中的高效表达及活性检测

为表达和纯化黄粉甲Tenebrio molitor抗冻蛋白, 采用RT-PCR方法扩增得到黄粉甲抗冻蛋白基因afp84a cDNA, 将其连接到pMAL-p2X质粒上, 构建分泌型融合表达载体pMAL-p2X-afp84a, 并在大肠杆菌Escherichia coli TBI中表达; 进一步利用Amylose柱亲和纯化出该重组蛋白, 后利用细菌抗寒性检测重组蛋白的生物活性。结果显示: 融合蛋白含量占总可溶蛋白的40%。SDS-PAGE分析表明, 用MgSO4处理法与超声波细胞破碎法均可使融合蛋白从细胞中释放; 融合蛋白经Amylose柱亲和纯化, Factor Xa因子酶切, 电泳显示获得的目的蛋白呈单一条带。细菌抗寒性检测表明该重组蛋白具有较高的抗冻活性。黄粉甲抗冻蛋白基因afp84a cDNA的克隆、 原核表达为进一步研究抗冻蛋白的性质和应用提供了有用的实验材料。     

Structure-function relationships in the tryptophan-rich, antimicrobial peptide indolicidin.

Indolicidin is a cationic 13 amino acid peptide amide produced in the granules of bovine neutrophils with the sequence H-ILPWKWPWWPWRR-NH2. Indolicidin is both antimicrobial and, to a lesser extent, haemolytic. In order to systematically investigate structure-function relationships, the solid-phase synthesis of indolicidin and 48 distinct analogues are reported, as well as the characterization of their respective biological properties. Peptides synthesized and characterized include analogues with modified terminal functions, truncations from either terminus, an alanine scan to determine the role of each individual amino acid, specific amino acid exchanges of aromatic, charged and structural residues and several retro-, inverso- and retroinverso-analogues. Together, characterization of these analogues identifies specific residues involved in antimicrobial or haemolytic activity and suggests a core structure that may form a scaffold for the further development of peptidomimetic analogues of indolicidin.     

Cyfluthrin and Deltamethrin Induce Changes in the Fat Body Composition of Tenebrio molitor Larvae,Males and Females

The purpose of present study was to analyze the chemical composition of the fat body of insects (Tenebrio molitor) and its eventual variation after the application of insecticides. Using the GC/MS technique, the fat body composition of larvae, males and females was analyzed. The insects were stressed with selected insecticides containing cyfluthrin (Cyflok 50EW) and deltamethrin (K‐othrine) recommended for this species of insect by insecticides producers and farmers. The effect of these two chemicals on the chemical compounds patterns from fat body of insects was compared. Possible differences in the fat body composition between developmental stages and the sex of insects were also compared. Possible identified compounds included amino acids, carboxylic acids, alcohols, sterols, glycerol and urea. Our results show that the composition of lipids was different depending on the stage of development, the insecticide used and the elapsed time since its application.     

Pleiotropic effects of the neuropeptides CCAP and myosuppressin in the beetle, Tenebrio molitor L.

Biological activities of crustacean cardioactive peptide (CCAP; PFCNAFTGCa) and leucomyosuppressin (Lem-MS; pQDVDHVFLRFa) were studied in heterologous bioassays in the larvae and adults of Tenebrio molitor. CCAP exerted a reversible and dose-dependent cardio-stimulatory effect on the semi-isolated heart of the experimental beetles at a concentration of >10(-7) M and induced an effect similar to the endogenic cardio-stimulatory peptide, proctolin. Injections of CCAP (10(-9)-10(-3) M) into 4-day-old adult reproductive females increased the concentration of soluble proteins in hemolymph in comparison to the saline injected controls. Electrophoretic analyses indicated significant increase in the level of two proteins 130 and 170 kDa, and a partial increase of the level of 67-kDa protein. The studies indicated that CCAP increased also free hemolymph sugar concentration in young larvae and adults of the mealworm beetle. The cardio-inhibitory peptide Lem-MS exerted the opposite effect: at concentration 10(-7)-10(-6) M, it significantly decreased the heartbeat frequency. The induced changes were dose-dependent and reversible, but at higher concentrations (>10(-5) M) the stimulatory effect disappeared. Injections of the Lem-MS into young larvae at concentrations 10(-9)-10(-3) M, also increased the free hemolymph sugar level similarly to the CCAP. This work demonstrates the pleiotropic effects of CCAP and Lem-MS in Tenebrio molitor.     

SPME‐GC/MS Analysis of Volatile Compounds Contained in the Insect Larvae of Tenebrio molitor and Leptinotarsa decemlineata before and after Using Insecticides

The larvae of two harmful insect species Tenebrio molitor and Leptinotarsa decemlineata were analyzed. The insects were sprayed with insecticides containing the active substances cyfluthrin and deltamethrin (T. molitor), and thiamethoxam and acetamiprid (L. decemlineata). The sprayed insect larvae were left for 24, 48 and 72 h. Samples were then prepared using SPME fiber to identify the volatile compounds contained in the larvae. The determinations were made by gas chromatography coupled with mass spectrometry (GC/MS). Chemical compounds were found in the analyzed samples: alkanes, aldehydes, ketones, esters, terpenes, fatty acids. In the samples prepared from T. molitor larvae, more than 20 % of aldehydes and 8–41 % of alkanes were determined. In the samples from L. decemlineata, fatty acids were 8–65 % depending on the length of time after applying the insecticide.     

Effects of targeting eye color in Tenebrio molitor through RNA interference of tryptophan 2,3‐dioxygenase (vermilion): Implications for insect farming

The gene vermilion encodes tryptophan 2,3‐dioxygenase, part of the ommochrome pathway, and is responsible for the dark pigmented eyes in some insects, including beetles. Using RNA interference, we targeted the vermilion gene ortholog in embryos and pupae of the yellow mealworm, Tenebrio molitor, resulting in larvae and adults, respectively, that lacked eye pigment. RNA‐Seq was used to analyze the impact of vermilion‐specific RNA interference on gene expression. There was a 425‐fold reduction in vermilion gene expression (p = 0.0003), as well as significant (p < 0.05) differential expression of 109 other putative genes, most of which were downregulated. Enrichment analysis of Gene Ontology terms found in the differentially expressed data set included genes known to be involved in the ommochrome pathway. However, enrichment analysis also revealed the influence of vermilion expression on genes involved in protein translocation to the endoplasmic reticulum, signal transduction, G‐protein‐coupled receptor signaling, cell‐cycle arrest, mannose biosynthesis, and vitamin transport. These data demonstrate that knockdown of vermilion in T. molitor results in complete loss of eye color (white‐eyed phenotype) and identify other interrelated genes in the vermilion metabolic pathway. Therefore, a dominant marker system based on eye color can be developed for the genetic manipulation of T. molitor to increase the value of mealworms as an alternative food source by decreasing negative traits, such as disease susceptibility, and increasing desired traits, such as protein content and vitamin production.     

3-(1',1'-Dimethylbutyl)-1-deoxy-delta8-THC and related compounds: synthesis of selective ligands for the CB2 receptor

The synthesis and pharmacology of 15 1-deoxy-delta8-THC analogues, several of which have high affinity for the CB2 receptor, are described. The deoxy cannabinoids include 1-deoxy-11-hydroxy-delta8-THC (5), 1-deoxy-delta8-THC (6), 1-deoxy-3-butyl-delta8-THC (7), 1-deoxy-3-hexyl-delta8-THC (8) and a series of 3-(1',1'-dimethylalkyl)-1-deoxy-delta8-THC analogues (2, n = 0-4, 6, 7, where n = the number of carbon atoms in the side chain-2). Three derivatives (17-19) of deoxynabilone (16) were also prepared. The affinities of each compound for the CB1 and CB2 receptors were determined employing previously described procedures. Five of the 3-(1',1'-dimethylalkyl)-1-deoxy-delta8-THC analogues (2, n = 1-5) have high affinity (Ki = < 20 nM) for the CB2 receptor. Four of them (2, n = 1-4) also have little affinity for the CB1 receptor (Ki = > 295 nM). 3-(1',1'-Dimethylbutyl)-1-deoxy-delta8-THC (2, n = 2) has very high affinity for the CB2 receptor (Ki = 3.4 +/- 1.0 nM) and little affinity for the CB1 receptor (Ki = 677 +/- 132 nM).     

Structure-antiviral activity relationships of cecropin A-magainin 2 hybrid peptide and its analogues.

In order to elucidate the structure-antiviral activity relationship of cecropin A (1-8)-magainin 2 (1-12) (termed CA-MA) hybrid peptide, several analogues with amino acid substitutions were synthesized. In a previous study, it was shown that serine at position 16 in CA-MA hybrid peptide was very important for antimicrobial activity. Analogues were designed to increase the hydrophobic property by substituting a hydrophobic amino acid residue (S --> A, V, F or W, position 16) in the CA-MA hybrid peptide. In this study, the structure-antiviral activity relationships of CA-MA and its analogues were investigated. In particular, substitution of Ser with a hydrophobic amino acid, Val, Phe or Trp at position 16 caused a dramatic increase in the virus-cell fusion inhibitory activity. These results suggested that the hydrophobicity at position 16 in the hydrophobic region of CA-MA is important for potent antiviral activity.     

Peptide dynamic fingerprints: a tool for investigating the role of conformational flexibility for GLP-1 analogs affinity.

Glucagon-like peptide-1 (GLP-1) is a 30-residue peptide implicated in short-term appetite regulation. Its analogs are presumed to be potential drugs against obesity and non-insulin dependent diabetes mellitus (NIDDM or type 2 diabetes). This study examined how the dynamic fingerprints can be used for establishing dynamics-activity relationships in a series of peptides for which the mechanism of action is unknown and in which mutations can cause an increase or decrease in biological activity. The 3D autocorrelation method was used to generate maps of both active and inactive analogs. As the active conformation of GLP-1 is not yet clearly defined, the dynamic fingerprints of peptides in an aqueous environment were compared to explain the high affinity of the peptide for its receptor. The suggestion that the peptide could bind to the receptor in a folded conformation has been examined. In the case of the GLP-1 analogs, it was shown that the folding tendency cannot be directly related to affinity values and the results do not favor a folded active conformation model of GLP-1.     

Synthesis and structure-activity relationship of beta-defensins, multi-functional peptides of the immune system.

beta-defensins are a large family of multiple disulfide-bonded peptides occurring in mammals and birds. They play an important role in the innate immune system, directly killing microbial organisms. Recent research has demonstrated that beta-defensins are important for other biological functions beyond antimicrobial effects, including inhibition of viral infection, interaction with Toll-like receptors, chemotactic effects, and sperm function. The corresponding broad spectrum of activities makes this peptide class an important subject and tool in immunologic research. In this review, we summarize the current status of the routes to obtain synthetic beta-defensins, their major structural properties and structure-activity relationship.