Quantitation of the insect electroantennogram: Measurement of sensillar contributions,elimination of background potentials,and relationship to olfactory sensation

A quantitative study of the electroantennogram (EAG) of Trichoplusia ni (Hübner) response to sex pheromone has provided new information about the physiological basis of the EAG and its relationship to behaviour. Procedures were developed that selectively stimulated defined numbers of sensilla and eliminated extraneous olfactory stimuli. The results show that the EAG is linearly proportional to the number of antennal sensilla stimulated and clarify interpretation of the blank response. Studies with calibrated dispensers indicate that the EAG is related to pheromone concentration by a power function frequently associated with quantitative measures of electrophysiological and behavioural responses in other animals. Based on correlations between the EAG, behaviour, and single-neurone responses, a model is presented that interrelates these three parameters as different indicators of a single quantitative process in the central nervous system.     

Orco mediates olfactory behaviors and winged morph differentiation induced by alarm pheromone in the grain aphid,Sitobion avenae

Olfaction is crucial for short distance host location and pheromone detection by insects. Complexes of olfactory receptors (ORs) are composed of odor-specific ORs and OR co-receptors (Orco). Orcos are widely co-expressed with odor-specific ORs and are conserved across insect taxa. A number of Orco orthologs have been studied to date, although none has been identified in cereal aphids. In this study, an Orco gene ortholog was cloned from the grain aphid, Sitobion avenae, and named “SaveOrco”; RNA interference (RNAi) reduced the expression of SaveOrco to 34.11% in aphids, resulting in weaker EAG (electroantennogram) responses to plant volatiles (Z-3-hexene-1-ol; methyl salicylate, MeSA) and aphid alarm pheromone (E-β-farnesene, EBF). Aphid wing differentiation induced by EBF was investigated in both RNAi treated and untreated aphids. EBF induced production of winged aphids in both pre-natal and post-natal periods in untreated aphids, but no such induction was observed in the RNAi-treated aphids. We conclude that SaveOrco is crucial for the aphid's response to pheromones and other volatiles, and is involved in wing differentiation triggered by EBF.     

被害马尾松（Pinus massoniana）针叶挥发性物质的提取、鉴定及蚕饰腹寄蝇（Blepharipa zebina）的电生理活性

试验使用动态顶空吸附的方法,提取了被害马尾松(Pinus massoniana)针叶挥发物,并通过气相色谱-质谱联用仪(GC-MS)及标准样品进行鉴定。结果表明,被害马尾松针叶挥发性物质主要包括绿叶挥发物、单萜、含氧单萜及倍半萜等四大类物质;挥发物中以单萜的含量最高,其它3类物质含量较低。通过触角电位图(EAG)技术,测定了23种挥发性物质标准样品的石蜡油溶液对蚕饰腹寄蝇(Blepharipa zebina)雌蝇的电生理活性。结果表明,蚕饰腹寄蝇雌蝇对α-蒎烯、β-蒎烯、长叶烯、莰烯、乙酸冰片酯等的相对触角电位反应值与湿润空气对照相比并无显著差异;雌蝇对其它刺激物的相对触角电位反应值与湿润空气对照相比均存在显著差异。同时测定了雌蝇对8种标样,在5个浓度系列下的剂量-反应,并分析了反应阈值。结果表明,蚕饰腹寄蝇雌蝇对顺-3-己烯-1-醇和里哪醇的反应阈值为0.1μg/μl,对壬醛的反应阈值为1μg/μl,对柠檬烯、α-水芹烯、2,6-二甲基-2,4,6-辛三烯、β-石竹烯4种物质的反应阈值为100μg/μl,而对α-蒎烯,在测试的剂量范围内不存在反应阈值。在确定对蚕饰腹寄蝇有生理活性的物质成分和分析反应阈值的基础上,对寄生蝇类定位寄主的机制进行了讨论。     

土壤中外源锌对不同植物毒性的敏感性分布

采用逻辑斯蒂克分布(log-logistic distribution)模型结合物种敏感性分布方程Burr-III分析,研究了2种不同土壤中添加不同水平的外源Zn后对8种不同植物毒性的剂量-效应关系及不同植物对外源Zn毒害的敏感性差异。结果表明,土壤中添加低浓度(<100mg·kg-1)Zn能对植物生长产生刺激效应,而过量的Zn则产生明显毒害作用。土壤中Zn毒性的阈值浓度(ECx)在不同植物间有较大差异,这主要与植物种类及土壤性质差异有关。不同土壤中Zn植物毒性的敏感性分布结果表明,不同植物对Zn毒性的敏感性频次分布有明显差异,其中叶菜类植物对土壤中Zn的毒害较为敏感,而禾本科类植物(如玉米)对Zn具有较强的抗性,不同类型植物对土壤中Zn毒害的敏感性分布频次顺序与土壤性质无关。     

Galvanotaxis of human granulocytes

The galvanotactic response of human granulocytes was investigated theoretically and experimentally. The basic results are: (i) The granulocytes move towards the anode. (ii) The directed movement has been quantified by two different polar order parameters-the McCutcheon index and the average of cos . (iii) The polar order parameters are a function of the applied electric field (= dose-response curve). (iv) The inverse of the galvanotactic constant of migrating cells (analogous to the Michaelis-Menten constant) has a value of-0.2±0.03 V/mm. (v) The galvanotactic response of granulocytes is a non-cooperative process with a cooperativity coefficient of 1±0.2. (vi) The galvanotactic constant is a function of pH. (vii) The protein essential for the galvanotactic response is very likely a G-protein.     

Increased EAG responses of tortricid moths after prolonged exposure to plant volatiles: evidence for octopamine-mediated sensitization

As measured by electroantennograms (EAG), both male and female obliquebanded leafrollers, Choristoneura rosaceana (Harris), and redbanded leafrollers, Argyrotaeniavelutinana (Walker), were similarly sensitive to host-related plant volatiles: trans-2-hexenal, benzaldehyde, 1-hexenol, cis-3-hexen-1-ol, geraniol, linalool, (+)-limonene, hexenal and trans-2-hexenol. Females of both species were similarly sensitive to the shared major component of their sex-attractant pheromone ((Z)11-14:Ac). Continuous 60 min pre-exposure of male and female C. rosaceana and A. velutinana to successively higher concentrations of a mixture of the nine plant volatiles in Teflon chambers with continuous air exchange caused a dosage-dependent increase in subsequent responsiveness (sensitization) to green leaf volatiles, as measured by EAGs. In addition, 60 min of pre-exposure of male C. rosaceana to certain individual volatiles ((+)-limonene, geraniol, benzaldehyde) increased EAGs nearly as much as did the mixture of nine volatiles. Pre-exposures to the nine plant-volatile mixture at concentrations achieved by 100 microg and 1 mg loading dosages in 100 microl of mineral oil significantly increased EAG depolarization to pheromone (cross-sensitization) in males but not females of both moth species. Antennae of male C. rosaceana pre-injected with 100 microg of octopamine (OA) without volatile pre-exposure exhibited sensitization nearly identical to that induced by pre-exposing moths to sensitizing concentrations of the plant-volatile mixture. Moreover, injection of the OA antagonist chlorpromazine (CP) blocked sensitization by the plant-volatile pre-exposure. Collectively, these findings suggest that exposures of tortricid moths to certain host-plant related volatiles may modulate subsequent olfactory sensitivity to behaviorally relevant chemical cues and that plant-volatile induced sensitization may be octopamine mediated.     

Melatonin entrains free-running blind people according to a physiological dose-response curve

The specific circadian role proposed for endogenous melatonin production was based on a study of sighted people who took low pharmacological doses (500 µg) of this chemical signal for the “biological night”: the magnitude and direction of the induced phase shifts were dependent on what time of day exogenous melatonin was administered and were described by a phase-response curve that turned out to be the opposite of that for light. We now report that lower (physiological) doses of up to 300 µg can entrain (synchronize) free-running circadian rhythms of 10 totally blind subjects that would otherwise drift later each day. The resulting log-linear dose-response curve in the physiological range adds support for a circadian function of endogenous melatonin in humans. Efficacy of exogenous doses in the physiological range are of clinical significance for totally blind people who will need to take melatonin daily over their entire lifetimes in order to remain entrained to the 24 h day. Left untreated, their free-running endocrine, metabolic, behavioral, and sleep/wake cycles can be almost as burdensome as not having vision.     

Electrophysiological characterisation of olfactory sensilla in the black bean aphid, Aphis fabae

Electrophysiological responses of three different olfactory sensilla (proximal primary rhinaria (PPR), distal primary rhinaria (DPR) and secondary rhinaria (SR)) to the sex pheromone components, nepetalactol, nepetalactone, and a plant volatile, (E)-2-hexenal, were investigated in four different morphs of the black bean aphid, Aphis fabae Scopoli. The DC responses recorded directly from the antennal sensilla and termed electrosensillograms (ESGs) were generally much larger (up to 14 mV) than electroantennogram (EAG) responses (up to 2.5 mV). Characteristic morph-specific response profiles to these compounds were observed in each type of rhinarium and response waveforms were different between (E)-2-hexenal and the sex pheromone components. (E)-2-Hexenal elicited the largest responses at PPR, while nepetalactol and nepetalactone elicited the largest responses at SR in gynoparae and males. Nepetalactol and nepetalactone also showed significant activities on DPR and PPR in all morphs. In contrast, (E)-2-hexenal had almost no activity on SR. However, almost all of the SR investigated in males and gynoparae were sensitive to both nepetalactol and nepetalactone. A small sub-set of male SR responded mainly to nepetalactol. SR of winged virginoparae did not respond to the sex pheromone compounds. Paraffin oil (the solvent control) also elicited significant responses at PPR in virginoparae but not in other morphs. In a further experiment, SR of winged virginoparae showed no response to 30 other plant volatile compounds or the alarm pheromone component, (E)-beta-farnesene. Nepetalactol and nepetalactone had similar dose-response profiles in the SR of gynoparae. The results indicate that SR in males and gynoparae are highly specialised to detect sex pheromone compounds, while the DPR and PPR are relatively broadly tuned to both plant volatiles and sex pheromone components. The presence of SR in winged virginoparae that are not responsive to sex pheromone components, alarm pheromone, or any of the plant volatile compounds tested may indicate a possible role of these sensilla to detect, as yet, unknown compound(s) with a high specificity. The present study also suggests that PPR may play a role in detecting paraffin-related compounds such as cuticular hydrocarbons of plants.     

Identification of a single killer immunoglobulin-like receptor (KIR) gene in the porcine leukocyte receptor complex on chromosome 6q

Human killer immunoglobulin-like receptors (KIR) are expressed on natural killer (NK) cells and are involved in their immunoreactivity. While KIR with a long cytoplasmic tail deliver an inhibitory signal when bound to their respective major histocompatibility complex class I ligands, KIR with a short cytoplasmic tail can activate NK responses. The expansion of the KIR gene family originally appeared to be a phenomenon restricted to primates (human, apes, and monkeys) in comparison to rodents, which via convergent evolution have numerous C-type lectin-like Ly49 molecules that function analogously. Further studies have shown that multiple KIR are also present in cow and horse. In this study, we have identified by comparative genomics the first and possibly only KIR gene, named KIR2DL1, in the domesticated pig (Sus scrofa) allowing further evolutionary comparisons to be made. It encodes a protein with two extracellular immunoglobulin domains (D0 + D2), and a long cytoplasmic tail containing two inhibitory motifs. We have mapped the pig KIR2DL1 gene to chromosome 6q. Flanked by LILRa, LILRb, and LILRc, members of the leukocyte immunoglobulin-like receptor (LILR) family, on the centromeric end, and FCAR, NCR1, NALP7, NALP2, and GP6 on the telomeric end, pig demonstrates conservation of synteny with the human leukocyte receptor complex (LRC). Both the porcine KIR and LILR genes have diverged sufficiently to no longer be clearly orthologous with known human LRC family members.     

黄秋葵挥发油对小菜蛾的触角电位反应及趋性研究

通过触角电位仪测定了小菜蛾Plutella xylostella(L.)对黄秋葵(Abelmoschus esculentus(L.)Moench Meth)挥发油的触角电位反应。结果表明,黄秋葵挥发油对小菜蛾成虫的触角电位反应强度与挥发油剂量成正比。四臂嗅觉仪实验进一步表明,黄秋葵挥发油对小菜蛾起到驱避作用。     

Modulation of Glucagon Receptor Pharmacology by Receptor Activity-modifying Protein-2 (RAMP2)

The glucagon and glucagon-like peptide-1 (GLP-1) receptors play important, opposing roles in regulating blood glucose levels. Consequently, these receptors have been identified as targets for novel diabetes treatments. However, drugs acting at the GLP-1 receptor, although having clinical efficacy, have been associated with severe adverse side-effects, and targeting of the glucagon receptor has yet to be successful. Here we use a combination of yeast reporter assays and mammalian systems to provide a more complete understanding of glucagon receptor signaling, considering the effect of multiple ligands, association with the receptor-interacting protein receptor activity-modifying protein-2 (RAMP2), and the role of individual G protein α-subunits. We demonstrate that RAMP2 alters both ligand selectivity and G protein preference of the glucagon receptor. Importantly, we also uncover novel cross-reactivity of therapeutically used GLP-1 receptor ligands at the glucagon receptor that is abolished by RAMP2 interaction. This study reveals the glucagon receptor as a previously unidentified target for GLP-1 receptor agonists and highlights a role for RAMP2 in regulating its pharmacology. Such previously unrecognized functions of RAMPs highlight the need to consider all receptor-interacting proteins in future drug development.     

Identification by GC-EAD of the two-component trail-following pheromone of Prorhinotermes simplex (Isoptera, Rhinotermitidae, Prorhinotermitinae)

GC/MS analysis confirmed that neocembrene is the major component of the trail pheromone in the three species of the termite genus Prorhinotermes (P. simplex, P. canalifrons, P. inopinatus). In addition, EAG and GC-EAD experiments with P. simplex strongly suggest that dodecatrienol is a quantitatively minor component but a qualitatively important component of this trail pheromone. Trail-following bioassays confirmed the two-component nature of the trail pheromone. This is the first report of the use of the GC-EAD for the identification of trail pheromone in termites. These original results underline once again the special phylogenetic status of the Prorhinotermitinae among Rhinotermitidae.     

Olfactory attraction to aggregation pheromone is mediated by disti-flagellum of antennal segments in Riptortus pedestris

Riptortus pedestris (Hemiptera: Alydidae) is a serious pest of soybean and sweet persimmon and uses male produced aggregation pheromone, (E)-2-hexenyl (Z)-3-hexenoate, (E)-2-hexenyl (E)-2-hexenoate, and tetradecyl isobutyrate to facilitate food location and recognition by conspecifics. Using electroantennogram (EAG) and greenhouse bioassay, we determined which antennal segment is involved in the detection of their aggregation pheromone. In the first EAG test using individual antennal segments, significant EAG responses to 1:1:1 mixture of the aggregation pheromone were observed only from the disti-flagellum segments of both male and female antennae at both pheromone doses tested (1 µg and 100 µg). In the following EAG tests using gradually removed antennal segment(s), EAG response was still maintained when the distal half of a disti-flagellum was surgically removed, while EAG response was lost when whole segment of disti-flagellum or other whole segments were gradually removed from intact antenna of both sexes. In greenhouse experiment, removing one or both segment(s) of disti-flagellum from male or female antennae resulted in significant reduction in their attraction to the aggregation pheromone. Together, these findings support that the disti-flagellum of R. pedestris houses olfactory neurons associated with attraction to their aggregation pheromone.     

Improvement of signal-to-noise ratio in electroantennogram responses using multiple insect antennae

Using an array of insect antennae connected in series or in parallel, electroantennogram (EAG) responses and noise levels were investigated in an attempt to improve signal-to-noise (S/N) ratio and sensitivity. Both the EAG response amplitude and noise level increased when the antennae of male Helicoverpa zea (Lepidoptera: Noctuidae) were connected in series. Due to lower relative increase in noise level than EAG amplitude as the number of antennae increased, the S/N ratio was also significantly improved by the serial connection. As a result the sensitivity of EAG was improved by the serial connection, which showed ca. ten-fold improvement in the threshold detection levels compared with a single antenna when four antennae were connected in series. In contrast to the serial connection, there were no differences in EAG amplitudes and overall noise levels when different numbers of antennae were connected in parallel. When only large-amplitude noise was taken into account, however, the S/N ratio was somewhat improved by the parallel connection. The frequency of overall noise remained at the same level both in the serial and in the parallel connection. However, the frequency of the large-amplitude noise increased in serial connection but decreased in parallel connection. The present study clearly indicates that both the sensitivity and S/N ratio of the EAG biosensor could be significantly improved by using the multiple antennal connections.     

斜纹夜蛾多角体病毒包埋型病毒受体的鉴定

蔗糖密度梯度离心法提纯斜纹夜蛾核多角体病毒（＄Ｓｐｏｄｏｐｔｅｒａ　ｌｉｔｕｒａ　　ｍｕｌｔｉｎｕｃｌｅｏｃａｐｓｉｄ　ｎｕｃｌｅｏｐｏｌｙｈｄｒｏｖｉｒｕｓ,　ＳｐｌｔＭＮＰＶ）包埋型病毒粒子（ｐｏｌｙｈｅｄｒａ－ｄｅｒｉｖｅｄ　ｖｉｒｕｓ,ＰＤＶ）,以此病毒粒子作抗原,免疫家兔获得抗血清;用ＳＤＳ裂解缓冲液提取斜纹夜蛾幼虫中肠组织细胞总蛋白。采用病毒铺覆蛋白印迹技术（ＶＯＰＢＡ）,利用抗ＰＶＤ抗血清对病毒受体进行检测,结果表明斜纹夜蛾中肠细胞总蛋白中４０ｋＤ、７３ｋＤ、８５ｋＤ的三种蛋白能够结合ＰＤＶ。     

Characterization of Cu2+-binding modes in the prion protein by visible circular dichroism and multivariate curve resolution

Visible circular dichroism (CD) spectra from the copper(II) titration of the metal-binding region of the prion protein, residues 57-98, were analyzed using the self-modeling curve resolution method multivariate curve resolution-alternating least squares (MCR-ALS). MCR-ALS is a set of mathematical tools for estimating pure component spectra and composition profiles from mixture spectra. Model-free solutions (e.g., soft models) are produced under the assumption that pure component profiles should be nonnegative and unimodal. Optionally, equality constraints can be used when the concentration or spectrum of one or more species is known. MCR-ALS is well suited to complex biochemical systems such as the prion protein which binds multiple copper ions and thus gives rise to titration data consisting of several pure component spectra with overlapped or superimposed absorption bands. Our study reveals the number of binding modes used in the uptake of Cu²⁺ by the full metal-binding region of the prion protein and their relative concentration profiles throughout the titration. The presence of a non-CD active binding mode can also be inferred. We show that MCR-ALS analysis can be initialized using empirically generated or mathematically generated pure component spectra. The use of small model peptides allows us to correlate specific Cu²⁺-binding structures to the pure component spectra.     

Phorid fly, Pseudacteon tricuspis, response to alkylpyrazine analogs of a fire ant, Solenopsis invicta, alarm pheromone

The phorid fly, Pseudacteon tricuspis Borgmeier, is a parasitoid of the red imported fire ant, Solenopsis invicta Buren. This fly has been reported to use fire ant chemicals, specifically venom alkaloids and possibly alarm pheromone to locate its host. A recent study identified 2-ethyl-3,6-dimethyl pyrazine as a component of the alarm pheromone of S. invicta. To determine the possible involvement of this fire ant alarm pheromone component in mediating fire ant-phorid fly interactions, we tested electroantennogram (EAG) and behavioral responses of P. tricuspis females to the commercially available mixture of 2-ethyl-3,6-dimethyl pyrazine and its 3,5-dimethyl isomer, as well as six structurally related alkylpyrazine analogs at varying doses. Pseudacteon tricuspis females showed significant EAG response to 2-ethyl-3,6(or 5)-dimethyl pyrazine (herein referred to as pheromone-isomer) at all doses, 0.001-10 μg. Among the tested alkylpyrazine analogs, 2,3-diethyl-5-methyl pyrazine showed significant EAG activity at 0.1 and 1 μg. 2,3-dimethyl pyrazine also showed significant EAG activity at 0.1 μg. Results of four-choice olfactometer bioassays demonstrated significant attraction of P. tricuspis females to the pheromone-isomer (2-ethyl-3,6(or 5)-dimethyl pyrazine) at all tested doses (0.01, 0.1, 1 and 10 μg). The analogs, 2,3-diethyl-5-methyl pyrazine and 2,3-dimethyl pyrazine were significantly better than the control at the higher doses (0.1, 1 and 10 μg). The pheromone-isomer was significantly better than both analogs at two doses, 0.1 and 1 μg. These results confirm that the reported fire ant alarm pheromone component plays a role in mediating attraction of phorid flies to host workers. Venom alkaloids were previously shown to attract P. tricuspis; therefore, we propose that fire ant alarm pheromones may act in tandem or synergistically with venom alkaloids to attract phorid fly parasitoids to fire ant workers.