Feeding responses of the horsefly, Tabanus nigrovittatus, to physical factors, ATP analogues and blood fractions

ABSTRACT. Wild-caught female horseflies, Tabanus nigrovittatus Macq. (Diptera: Tabanidae), were presented solutions of seven analogues of ATP in 0.15 m NaCl, or various blood fractions, either as free liquids at 22 or 38C or covered with a Parafilm M membrane at 38C. Warming the diet, so that it can stimulate the insects' heat receptors, or presenting it warmed and covered with a membrane, which the flies can pierce and thus deploy their mouthparts as they would when blood-feeding, enhances the response to gorging stimulants. ADP (ED₅₀ 45 μM) was the most potent chemical phagostimulant. There were no significant differences between the potencies of AMP, A(TETRA)P, AMP-PCP, AMP-PNP or AMP-S, which were 3.5-5 times less potent than ADP. Cyclic AMP had no phagostimulatory activity at concentrations of 400 or 1000 μM. The ED₅₀ for washed red blood cells (RBC) in saline was 4.5% (one tenth the concentration found in blood). RBC-free plasma caused only 10% gorging but plasma with 0.5% RBC caused 61% gorging, indicating synergism between RBC and plasma.     

Feeding behaviour of the horsefly Tabanus nigrovittatus (Diptera: Tabanidae): effects of dissolved solids on ingestion and destination of sucrose or ATP diets

Abstract Wild caught female horseflies, Tabanus nigrovittatus Macq. (Diptera: Tabanidae), were fed solutions containing either sucrose or ATP as a phagostimulant. The sucrose diets were presented as free liquids at room temperature. The ATP in the other diets was dissolved in 0.15 M NaCl, or in 0.3 M or 1 M cellobiose and was presented covered with a Parafilm membrane at 38^oC. The content of dissolved solids in some diets was adjusted with added cellobiose, which had no phago-stimulatory effect. Diets of 1 M or 0.6 M sucrose were ingested in large amounts and were directed to the crop. Diets of 0.3 M or 0.1 M sucrose induced lower levels of ingestion, and the diet was directed to the midgut. The addition of cellobiose to give a 1 M total concentration of dissolved solids in these sucrose diets induced many insects responding to the lower concentrations of sucrose to direct the diet to the crop. Diets of ATP in saline or in 0.3 M cellobiose were directed to the midgut. ATP in 1 M cellobiose caused many of the insects to put the diet in both the crop and the midgut. Two behavioural models are presented that integrate interactions between the level of phagostimulation, the concentration of dissolved solids, and an endogenous feeding factor. The dissolved solids effect is most likely a response to osmotic pressure.     

Feeding behaviour in response to blood fractions and chemical phagostimulants in the black-fly, Simulium venustum

ABSTRACT. Wild-caught black-flies ( Simulium venustum Say complex) were presented with diets at 37°C in an artificial feeding apparatus. Washed human red cells resuspended 1:1 in Ringer solution were potent phagostimulants, causing 89% of flies to gorge. Whole plasma was more potent (32% gorging) than platelet-poor plasma (2%). The ED₅₀ for red cells was 3.5%. Although ADP, contained in high-concentration in platelets, was confirmed as a more potent phagostimulant than ATP (ED₅₀ of 5πM V. 12μM), red cells were clearly more phagostimulatory than platelets, and with a potency more than adequate to trigger gorging in vivo. A high response to the ATP analogues, β, γ-methylene ATP and adenine phosphosulphate, supports the view that the phosphate chain is relatively unimportant in determining nucleotide potency to simuliids. The compounds phytic acid and 2,3-disphosphoglycerate, potent stimulants to Rhodnius prolixus , produced only moderate and no response, respectively at 1 mM; 5-hydroxytryptamine, another major constituent of platelets, also produced only a moderate response. Only flies caught while showing a characteristic probing behaviour would subsequently probe and feed in vitro; this 'blood-feeding mode' rapidly disappeared in the absence of stimuli eliciting actual probing, but for flies in this state a small temperature rise was sufficient stimulus for probing.     

Taxonomic Revision of Tabanus mandarinus Species Group (Diptera: Tabanidae) from Korea

ABSTRACT In a review of the horseflies of the Tabanus mandarinus species group in Korea, six species are recognized. Among them, Tabanus nipponicus is newly recorded from Korea. Keys, annotated check lists of domestic records, collection data, and photographs of T. nipponicus are provided.     

Feeding strategies of the horseflies Hybomitra expollicata and Tabanus bromius in southern France

Blood-feeding success of female horseflies, Hybomitra expollicata Pandellé and Tabanus bromius L. (Diptera: Tabanidae), was studied. Bloodmeal weights of flies permitted different periods of residence on a horse's belly were estimated, and related to subsequent ovarian development and the number of mature eggs produced. Fitness gain per visit to a host (as estimated by the number of mature eggs produced) increased more rapidly with residence time on the host for H.expollicata than for T.bromius. H.expollicata inhibed blood more rapidly and, above a threshold bloodmeal size, produced a consistently higher number of eggs per unit weight of blood ingested. The feeding strategies of the two species are compared.     

Ingestion and diet destination in Culiseta inornata: responses to water, sucrose and cellobiose

ABSTRACT. At ambient temperature, contact with free liquids containing sugar induces Culiseta inornata Williston (Diptera: Culicidae) females to adopt the nectar-feeding mode of behaviour. While in this mode, two distinct control mechanisms appear to operate: one controlling the amount of ingestion, the other diet destination. Mosquitoes were exposed to water and various concentrations of sucrose and cellobiose in water. Only small or taste amounts of water were imbibed, and it was directed to the midgut. Cellobiose solutions of less than 0.1M were treated as water. Cellobiose was phagostimulatory; as the concentration increased from 0.1 to 0.5 M the percentage of insects ingesting large amounts rose from 11% to 68%. Cellobiose diets were directed primarily to the midgut. Sucrose was also phagostimulatory. As the concentration increased from 0.025 to 0.2 M, the percentage of insects ingesting large amounts increased from 20 to 92. At lower sucrose levels, insects ingesting large amounts put it mainly into the crop, while insects ingesting smaller amounts put the diet into the midgut or both the crop and midgut. At sucrose concentrations of 0.4 M or more, most insects took large amounts and directed it to the crop. Different combinations of sucrose and cellobiose with a total concentration of 0.5 M all caused insects to take mainly large meals. At sucrose concentrations of 0.2M and above, the insects responded as they would to sucrose alone, directing the diet to the crop. At sucrose concentrations of 0.1M or less, diet destination patterns were more complex, with individual animals depositing the diet in the crop, the midgut, or in both. A behavioural model is presented which postulates different receptors for sucrose and cellobiose, control of diet destination via differential control of midgut and crop valves, and variation of threshold values according to endogenous factors.     

The oviduct musculature of the horsefly, Tabanus sulcifrons, and its response to 5-hydroxytryptamine and proctolin

ABSTRACT. A delicate lace-like membrane covers the ovaries of Tabanus sulcifrons. These membranes were found to contain muscle fibres that provide the organs with motile properties. The lateral oviducts consist of a single layer of longitudinal muscles that form a structural syncytium by means of an extensive anastomosis of the fibres comprising it. The common oviduct is composed of two muscle layers, an outer sheath of circular muscle and an inner substratum of longitudinal muscle. Both of these layers showed evidence of a structural syncytium. When isolated in saline, the oviduct was spontaneously active and gave a simple phasic pattern of contraction. Such muscle preparations were sensitive to both 5-hydroxytryptamine (5HT) and the insect myotropic peptide, proctolin. Excitation was generally indicated by a rise in muscle tonus or an increase in the frequency and amplitude of individual phasic contractions, or all three characteristics. The threshold for activation with 5HT was variable, ranging from as low as 4 × 10^-9M to 1 × 10^-7M. Proctolin evoked a noticeable increase in the tonus of most oviducts at 10^-10M. However, several preparations responded to as little as 3 × 10^-11M proctolin.     

华广虻(Tabanusamaenus Walker)溶纤活性蛋白的纯化及生物活性分析

经过 ７５％ 饱和度硫酸铵沉淀、 Ｓｅｐｈａｄｅｘ Ｇ ７５ 凝胶过滤层析、 Ｌｙｓ Ｓｅｐｈａｒｏｓｅ ４ Ｂ 亲和层析和电泳制备洗脱,从华广虻（ Ｔａｂａｎｕｓ ａｍ ａｅｎｕｓ Ｗ ａｌｋｅｒ）腹部组织匀浆液中分离纯化出分子量约为 ６７ｋ Ｄ 的溶纤活性蛋白 Ｔ Ａ Ｆ Ｐ经纤维蛋白平板测定表明, Ｔ Ａ Ｆ Ｐ 只具有纤溶酶作用,不具有激活纤溶酶原的作用;但 Ｔ Ａ Ｆ Ｐ 能分解纤溶酶原激活剂的生色底物—— Ｃｈｒｏｍ ｏｚｙｍ Ｕ Ｋ 及 Ｓ ２２８８还能水解胰蛋白酶专一底物 Ｂｚ Ｐｈｅ Ｖａｌ Ａｒｇ Ｎ Ａ 及 Ｃ Ｂ Ｚ Ｇｌｙ Ｐｒｏ Ａｒｇ Ｎ Ａ,表明 Ｔ Ａ Ｆ Ｐ具有类胰蛋白酶活性,专一水解精氨酸形成的酰胺键（或肽键） Ｔ Ａ Ｆ Ｐ无胰凝乳蛋白酶活性      

Chemical,immunological, enzymatic,and genetic approaches to studying the arrangement of the peptide chain of the ADP/ATP carrier in the mitochondrial membrane

In the process of oxidative phosphorylation, the exchange of cytosolic ADP^3– against mitochondrial ATP^4– across the inner mitochondrial membrane is mediated by a specific carrier protein. Two different conformations for this carrier have been demonstrated on the basis of interactions with specific inhibitors, namely carboxyatractyloside (CATR) and bongkrekic acid (BA). The two conformations, referred to as CATR and BA conformations, are interconvertible, provided that ADP or ATP are present. The functional ADP/ATP carrier is probably organized as a tetramer. In the presence of CATR or BA the tetramer is split into two dimers combined with either of the two inhibitors. The amino acid sequence of the beef heart carrier monomer (297 residues) contains three repeats of about 100 residues each. Experimental results obtained through different approaches, including photolabeling, immunochemistry, and limited proteolysis, can be interpreted on the basis of a model with five or six transmembrane helices per carrier monomer. Two mobile regions involved in the binding of nucleotides and accessible to proteolytic enzymes have been identified. Each of them may be visualized as consisting of two pairs of short amphipathic helices, which can be juxtaposed to form hydrophilic channels facilitating the nucleotide transport. Mutagenesis in yeast is currently being used to detect strategic amino acids in ADP/ATP transport.     

ATP-levels of germinating seeds in relation to vigor

Determination of seed vigor was attempted by comparing ATP-levels of deteriorating seed to germination percentage and production of dry matter. Immediately after imbibition of any seed lot investigated, a production of ATP took place. This ATP-accumulation invariably reached a plateau after 6 h of imbibition. Two well germinating seed lots of rape, one of cauliflower and one of sugar beet, were artificially aged by means of elevated storage temperature and humidity. Every second week through 16 weeks of deterioration the levels of ATP, ADP and AMP after 7 h of imbibition were compared with the germination percentage. While ADP- and AMP-contents of germinating seed displayed no change (when imbibed 7 h) during the period of artificial aging, seed deterioration was reflected in the ATP-levels long before loss of viability could be detected by the conventional germination test.
When ATP-levels per seed were related to germination percentage throughout the aging, all four seed lots displayed similar patterns although the absolute figures differed. In contrast to the conventional "per seed' basis, however, ATP per gram seed not only displayed similar deterioration patterns, but the absolute values were also of the same magnitude.     

Engorgement response of anopheline mosquitoes to blood fractions and artificial solutions

ABSTRACT. Anopheles stephensi Liston, Anopheles freeborni Aitken, Anopheles gambiae Giles and Anopheles dirus (Peyton & Harrison) fed equally well on whole blood, red blood cells, platelet-rich plasma and platelet-poor plasma. Similar feeding ability on 0.15 M NaCl containing 10^-2 M NaHCO₃ was shown by the first three species, but An. dirus required an addition of albumin. The need for ATP as a phagostimulant could not be demonstrated in any of these species.     

Biotin enhances ATP synthesis in pancreatic islets of the rat, resulting in reinforcement of glucose-induced insulin secretion

Previous studies showed that biotin enhanced glucose-induced insulin secretion. Changes in the cytosolic ATP/ADP ratio in the pancreatic islets participate in the regulation of insulin secretion by glucose. In the present study we investigated whether biotin regulates the cytosolic ATP/ADP ratio in glucose-stimulated islets. When islets were stimulated with glucose plus biotin, the ATP/ADP ratio increased to approximately 160% of the ATP/ADP ratio in islets stimulated with glucose alone. The rate of glucose oxidation, assessed by CO(2) production, was also about 2-fold higher in islets treated with biotin. These increasing effects of biotin were proportional to the effects seen in insulin secretion. There are no previous reports of vitamins, such as biotin, directly affecting ATP synthesis. Our data indicate that biotin enhances ATP synthesis in islets following the increased rate of substrate oxidation in mitochondria and that, as a consequence of these events, glucose-induced insulin release is reinforced by biotin.     

Four mutations in transmembrane domains of the mitochondrial ADP/ATP carrier increase resistance to bongkrekic acid

Two distinct conformations of the mitochondrial ADP/ATP carrier involved in the adenine nucleotide transport are called BA and CATR conformations, as they were distinguished by binding of specific inhibitors bongkrekic acid (BA) and carboxyatractyloside (CATR), respectively. To find out which amino acids are implicated in the transition between these two conformations, which occurs during transport, mutants of the Saccharomyces cerevisiae ADP/ATP carrier Anc2p responsible for resistance of yeast cells to BA were identified and characterized after in vivo chemical or UV mutagenesis. Only four different mutations could be identified in spite of a large number of mutants analyzed. They are located in the Anc2p transmembrane segments I (G30S), II (Y97C), III (L142S), and VI (G298S), and are independently enabling growth of cells in the presence of BA. The variant and wild-type Anc2p were produced practically to the same level in mitochondria, as evidenced by immunochemical analysis and by atractyloside binding experiments. ADP/ATP exchange mediated by Anc2p variants in isolated mitochondria was more efficient than that of the wild-type Anc2p in the presence of BA, confirming that BA resistance of the mutant cells was linked to the functional properties of the modified ADP/ATP carrier. These results suggest that resistance to BA is caused by alternate conformation of Anc2p due to appearance of Ser or Cys at specific positions. Different interactions of these residues with other amino acids and/or BA could prevent formation of stable inactive Anc2p BA complex.     

Sensitivity of yeast glycolytic enzymes to chloroquine

Chloroquine at pH 8.0 and 10 mM concentration inhibits about 30% glucose consumption and ethanol formation in yeast cells. Out of the 11 glycolytic enzymes assayed, phosphoglycerate kinase and pyruvate decarboxylase have been found to be most sensitive to chloroquine. Next sensitive are hexokinase, glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase. Kinetic studies with the three kinases studied revealed competitive inhibition of chloroquine with ATP (hexokinase, phosphoglycerate kinase) or ADP (pyruvate kinase).     

Studies of the ADP/ATP carrier of mitochondria with fluorescent ADP analogue formycin diphosphate

The ADP/ATP carrier was studied by a fluorescent substrate, formycin diphosphate which is the only fluorescent ADP analogue to bind. Its low quantum yield, short decay time and spectral overlap with tryptophan has as yet prevented its wider use.By incorporating fluorescent acceptors of formycin diphosphate fluorescence, anthracene-maleimide and vinylanthracene, into the membrane, these difficulties were circumvented. Only bound formycin diphosphate transfers energy to the probes so that the secondary emission of these probes is a measure for membrane-bound formycin diphosphate.The fluorescent transfer is inhibited by ADP, bongkrekate and carboxy-atractylate whether added before or after incubation of formycin diphosphate showing that only binding to the adenine nucleotide carrier is measured. It also shows directly that the earlier demonstrated ADP fixation by bongkrekate is indeed a displacement into the matrix.The fluorescence decay time of the bound formycin diphosphate is measured as 1.95 ns compared to 0.95 ns of the free formycin diphosphate, indicating that formycin diphosphate is bound at the carrier in a non-polar environment.The depolarization decay time was found to be larger than 15 ns, indicating that carrier-bound formycin diphosphate is immobile within this time period.     

Regional Changes in the Cellular Level of Adenine Nucleotides in Ischemic Rat Brain Subjected to Single Embolization

Regional changes in adenine nucleotides in the rat brain were studied after 1 h of ischemia produced by the embolization method. The animals were divided into three groups according to neurological symptoms: sham-operation group, group A (hemiparesis only), and group B (hemiparesis with unconsciousness). Marked ATP depletion was detected in the hippocampus on the embolized side and extended to the other regions on the same side in group B. The results suggest that this damage in regional energy metabolism arises from regional reduction in blood flow and/or tissue vulnerability. ATP levels in the hypothalamus, hippocampus, and striatum on the opposite side of embolization decreased markedly in group B, and may be caused by extension of brain edema or diaschisis.     

Effect of xiphidiocercarial infection on the bioenergetic parameters of Lymnaea luteola

The levels of adenine compounds in the gastropod Lymnaea luteola have been determined through ion-exchange chromatography. In snails infected with the larvae of Prosthogonimus sp., the concentration decreased. As a result, the different bioenergetic parameters like ATP/ADP, ATP/AMP, and “energy charge” were altered.     

The delivery of ADP/ATP carrier protein to mitochondria probed by fusions with green fluorescent protein and beta-galactosidase

The import of proteins into mitochondria is an essential process, largely investigated in vitro with isolated mitochondria and radioactively labeled precursors. In this study, we used intact cells and fusions with genes encoding two reporter proteins, green fluorescent protein (GFP) and beta-galactosidase (lacZ), to probe the import of the ADP/ATP carrier (AAC). Typical mitochondrial fluorescence was observed with AAC-GFP fusions containing at least one complete transmembrane loop. This confirms the results of in vitro analysis demonstrating that an internal targeting signal was present in each one of the three transmembrane loops of the carrier. The fusions of AAC fragments to beta-galactosidase demonstrated that the targeting signal was capable of delivering the reporter molecule to the mitochondrial surface, but not to internalize it to a protease-inaccessible location. The delivery to a protease-inaccessible location required the presence of more distal sequences present within the third (C-terminal) transmembrane loop of the carrier molecule. The results of our study provide an alternative for investigation in a natural context of mitochondrial protein import in cells when the isolation of intact, functional mitochondria is not achievable.     

Fatty acid-promoted mitochondrial permeability transition by membrane depolarization and binding to the ADP/ATP carrier

The mechanism by which non-esterified long-chain fatty acids (FFA) promote mitochondrial permeability transition (MPT) is not clear. We examined with energized rat liver mitochondria the role of two possible actions of FFA in MPT, (i) the reduction of the transmembrane potential (Δψ) and (ii) the increase of the negative surface charge of the inner mitochondrial membrane [Broekemeier, K.M. and Pfeiffer, D.G., Biochemistry 43, (1995) 16440–16449]. It was found that the ability of FFA to stimulate large amplitude swelling is clearly related to their uncoupling activity. Moreover, compared with classical protonophores (FCCP) FFA increase the sensitivity of the pore opening process to Δψ changes. In addition, FFA interact like their thioester derivatives in a structure-dependent manner with the ADP/ATP carrier (measured as inhibition of [³H]atractyloside binding to the AAC protein). It is suggested that not only the protonophoric action of FFA, but also a presumable stabilization of the ‘cytosolic' conformation of AAC contribute to the FFA-promoted MPT.