Prospects for the classical biological control of Calotropis procera (Apocynaceae) using co-evolved insects

Calotropis procera (Apocynaceae), a native of tropical Africa, the Middle East and the Indian subcontinent, is a serious environmental and rangeland weed of Australia and Brazil. It is also a weed in Hawaii in USA, the Caribbean Islands, the Seychelles, Mexico, Thailand, Vietnam and many Pacific Islands. In the native range C. procera has many natural enemies, thus classical biological control could be the most cost-effective option for its long-term management. Based on field surveys in India and a literature search, some 65 species of insects and five species of mites have been documented on C. procera and another congeneric-invador C. gigantea in the native range. All the leaf-feeding and stem-boring agents recorded on Calotropis spp. have wide host range. Three pre-dispersal seed predators,the Aak weevil Paramecops farinosus and the Aak fruit fly Dacuspersicus in the Indian subcontinent, and the Sodom apple fruit fly Dacus longistylus in the Middle East have been identified as prospective biological control agents based on their field host range. In Australia and Brazil, where C. procera has the potential to spread across vast areas, pre-dispersal seed predators would help to limit the spread of the weed. While the fruits of C. procera vary in size and shape across its range, those from India are similar to the ones in Australia and Brazil. Hence, seed-feeding insects from India are more likely to be suitable due to adaptation to fruit size and morphology. Future survey efforts for potential biological control agents should focus on North Africa.

A generic individual-based spatially explicit model as a novel tool for investigating insect-plant interactions: A case study of the behavioural ecology of frugivorous Tephritidae

Computational modelling of mechanisms underlying processes in the real world can be of great value in understanding complex biological behaviours. Uptake in general biology and ecology has been rapid. However, it often requires specific data sets that are overly costly in time and resources to collect. The aim of the current study was to test whether a generic behavioural ecology model constructed using published data could give realistic outputs for individual species. An individual-based model was developed using the Pattern-Oriented Modelling (POM) strategy and protocol, based on behavioural rules associated with insect movement choices. Frugivorous Tephritidae (fruit flies) were chosen because of economic significance in global agriculture and the multiple published data sets available for a range of species. The Queensland fruit fly (Qfly), Bactrocera tryoni, was identified as a suitable individual species for testing. Plant canopies with modified architecture were used to run predictive simulations. A field study was then conducted to validate our model predictions on how plant architecture affects fruit flies’ behaviours. Characteristics of plant architecture such as different shapes, e.g., closed-canopy and vase-shaped, affected fly movement patterns and time spent on host fruit. The number of visits to host fruit also differed between the edge and centre in closed-canopy plants. Compared to plant architecture, host fruit has less contribution to effects on flies’ movement patterns. The results from this model, combined with our field study and published empirical data suggest that placing fly traps in the upper canopy at the edge should work best. Such a modelling approach allows rapid testing of ideas about organismal interactions with environmental substrates in silico rather than in vivo, to generate new perspectives. Using published data provides a saving in time and resources. Adjustments for specific questions can be achieved by refinement of parameters based on targeted experiments.

Classification of Myopathies on Molecular basis in Drosophila using Raman spectroscopy

Myopathies are muscular diseases in which muscle fibers degenerate due to many factors such as nutrient deficiency, infection and mutations in myofibrillar etc. The objective of this study is to identify the bio-markers to distinguish various muscle mutants in Drosophila (fruit fly) using Raman Spectroscopy. Principal Components based Linear Discriminant Analysis (PC-LDA) classification model yielding >95% accuracy was developed to classify such different mutants representing various myopathies according to their physiopathology.

Classification of Myopathies on Molecular basis in Drosophila using Raman spectroscopy

Myopathies are muscular diseases in which muscle fibers degenerate due to many factors such as nutrient deficiency, infection and mutations in myofibrillar etc. The objective of this study is to identify the bio-markers to distinguish various muscle mutants in Drosophila (fruit fly) using Raman Spectroscopy. Principal Components based Linear Discriminant Analysis (PC-LDA) classification model yielding >95% accuracy was developed to classify such different mutants representing various myopathies according to their physiopathology.

A non-resonant mass sensor to eliminate the ``missing mass'' effect during mass measurement of biological materials

Resonant sensors and crystal oscillators for mass detection need to be excited at very high natural frequencies (MHz). Use of such systems to measure mass of biological materials affects the accuracy of mass measurement due to their viscous and/or viscoelastic properties. The measurement limitation of such sensor system is the difficulty in accounting for the ``missing mass'' of the biological specimen in question. A sensor system has been developed in this work, to be operated in the stiffness controlled region at very low frequencies as compared to its fundamental natural frequency. The resulting reduction in the sensitivity due to non-resonant mode of operation of this sensor is compensated by the high resolution of the sensor. The mass of different aged drosophila melanogaster (fruit fly) is measured. The difference in its mass measurement during resonant mode of operation is also presented. That, viscosity effects do not affect the working of this non-resonant mass sensor is clearly established by direct comparison. (C) 2014 AIP Publishing LLC.

Dynamic response of a piezoelectric flapping wing

Piezo-composite membranes have advantages over motorized flapping where frequencies are high and certain coupling between bending and twisting is useful to generate lift and forward flight. We draw examples of fruit fly and bumble bee. Wings with Piezo ceramic PZT coating are realized. The passive mechanical response of the wing is characterized experimentally and validated using finite element simulation. Piezoelectric actuation with uniform electrode coating is characterized and optimal frequencies for flapping are identified. The experimental data are used in an empirical model and advanced ratio for a flapping insect like condition for various angular orientations is estimated.

Social Experience Is Sufficient to Modulate Sleep Need of Drosophila without Increasing Wakefulness

Organisms quickly learn about their surroundings and display synaptic plasticity which is thought to be critical for their survival. For example, fruit flies Drosophila melanogaster exposed to highly enriched social environment are found to show increased synaptic connections and a corresponding increase in sleep. Here we asked if social environment comprising a pair of same-sex individuals could enhance sleep in the participating individuals. To study this, we maintained individuals of D. melanogaster in same-sex pairs for a period of 1 to 4 days, and after separation, monitored sleep of the previously socialized and solitary individuals under similar conditions. Males maintained in pairs for 3 or more days were found to sleep significantly more during daytime and showed a tendency to fall asleep sooner as compared to solitary controls (both measures together are henceforth referred to as ``sleep-enhancement''). This sleep phenotype is not strain-specific as it is observed in males from three different ``wild type'' strains of D. melanogaster. Previous studies on social interaction mediated sleep-enhancement presumed `waking experience' during the interaction to be the primary underlying cause; however, we found sleep-enhancement to occur without any significant increase in wakefulness. Furthermore, while sleep-enhancement due to group-wise social interaction requires Pigment Dispersing Factor (PDF) positive neurons; PDF positive and CRYPTOCHROME (CRY) positive circadian clock neurons and the core circadian clock genes are not required for sleep-enhancement to occur when males interact in pairs. Pair-wise social interaction mediated sleep-enhancement requires dopamine and olfactory signaling, while visual and gustatory signaling systems seem to be dispensable. These results suggest that socialization alone (without any change in wakefulness) is sufficient to cause sleep-enhancement in fruit fly D. melanogaster males, and that its neuronal control is context-specific.

Flight dynamics in Drosophila through a dynamically-scaled robotic approach

Flies are particularly adept at balancing the competing demands of delay tolerance, performance, and robustness during flight, which invites thoughtful examination of their multimodal feedback architecture. This dissertation examines stabilization requirements for inner-loop feedback strategies in the flapping flight of Drosophila, the fruit fly, against the backdrop of sensorimotor transformations present in the animal. Flies have evolved multiple specializations to reduce sensorimotor latency, but sensory delay during flight is still significant on the timescale of body dynamics. I explored the effect of sensor delay on flight stability and performance for yaw turns using a dynamically-scaled robot equipped with a real-time feedback system that performed active turns in response to measured yaw torque. The results show a fundamental tradeoff between sensor delay and permissible feedback gain, and suggest that fast mechanosensory feedback provides a source of active damping that compliments that contributed by passive effects. Presented in the context of these findings, a control architecture whereby a haltere-mediated inner-loop proportional controller provides damping for slower visually-mediated feedback is consistent with tethered-flight measurements, free-flight observations, and engineering design principles. Additionally, I investigated how flies adjust stroke features to regulate and stabilize level forward flight. The results suggest that few changes to hovering kinematics are actually required to meet steady-state lift and thrust requirements at different flight speeds, and the primary driver of equilibrium velocity is the aerodynamic pitch moment. This finding is consistent with prior hypotheses and observations regarding the relationship between body pitch and flight speed in fruit flies. The results also show that the dynamics may be stabilized with additional pitch damping, but the magnitude of required damping increases with flight speed. I posit that differences in stroke deviation between the upstroke and downstroke might play a critical role in this stabilization. Fast mechanosensory feedback of the pitch rate could enable active damping, which would inherently exhibit gain scheduling with flight speed if pitch torque is regulated by adjusting stroke deviation. Such a control scheme would provide an elegant solution for flight stabilization across a wide range of flight speeds.

Octopamine neurons mediate flight-induced modulation of visual processing in Drosophila melanogaster

Activity-dependent modulation of sensory systems has been documented in many organisms, and is likely to be essential for appropriate processing of information during different behavioral states. However, the mechanisms underlying these phenomena, and often their functional consequences, remain poorly characterized. I investigated the role of octopamine neurons in the flight-dependent modulation observed in visual interneurons in the fruit fly Drosophila melanogaster. The vertical system (VS) cells exhibit a boost in their response to visual motion during flight compared to quiescence. Pharmacological application of octopamine evokes responses in quiescent flies that mimic those observed during flight, and octopamine neurons that project to the optic lobes increase in activity during flight. Using genetic tools to manipulate the activity of octopamine neurons, I find that they are both necessary and sufficient for the flight-induced visual boost. This work provides the first evidence that endogenous release of octopamine is involved in state-dependent modulation of visual interneurons in flies. Further, I investigated the role of a single pair of octopamine neurons that project to the optic lobes, and found no evidence that chemical synaptic transmission via these neurons is necessary for the flight boost. However, I found some evidence that activation of these neurons may contribute to the flight boost. Wind stimuli alone are sufficient to generate transient increases in the VS cell response to motion vision, but result in no increase in baseline membrane potential. These results suggest that the flight boost originates not from a central command signal during flight, but from mechanosensory stimuli relayed via the octopamine system. Lastly, in an attempt to understand the functional consequences of the flight boost observed in visual interneurons, we measured the effect of inactivating octopamine neurons in freely flying flies. We found that flies whose octopamine neurons we silenced accelerate less than wild-type flies, consistent with the hypothesis that the flight boost we observe in VS cells is indicative of a gain control mechanism mediated by octopamine neurons. Together, this work serves as the basis for a mechanistic and functional understanding of octopaminergic modulation of vision in flying flies.

Complex behavior and perception in Drosophila emerges from iterative feedback-regulated reflexes

For a hungry fruit fly, locating and landing on a fermenting fruit where it can feed, find mates, and lay eggs, is an essential and difficult task requiring the integration of both olfactory and visual cues. Understanding how flies accomplish this will help provide a comprehensive ethological context for the expanding knowledge of their neural circuits involved in processing olfaction and vision, as well as inspire novel engineering solutions for control and estimation in computationally limited robotic applications. In this thesis, I use novel high throughput methods to develop a detailed overview of how flies track odor plumes, land, and regulate flight speed. Finally, I provide an example of how these insights can be applied to robotic applications to simplify complicated estimation problems. To localize an odor source, flies exhibit three iterative, reflex-driven behaviors. Upon encountering an attractive plume, flies increase their flight speed and turn upwind using visual cues. After losing the plume, flies begin zigzagging crosswind, again using visual cues to control their heading. After sensing an attractive odor, flies become more attracted to small visual features, which increases their chances of finding the plume source. Their changes in heading are largely controlled by open-loop maneuvers called saccades, which they direct towards and away from visual features. If a fly decides to land on an object, it begins to decelerate so as to maintain a stereotypical ratio of expansion to retinal size. Once they reach a stereotypical distance from the target, flies extend their legs in preparation for touchdown. Although it is unclear what cues they use to trigger this behavior, previous studies have indicated that it is likely under visual control. In Chapter 3, I use a nonlinear control theoretic analysis and robotic testbed to propose a novel and putative mechanism for how a fly might visually estimate distance by actively decelerating according to a visual control law. Throughout these behaviors, a common theme is the visual control of flight speed. Using genetic tools I show that the neuromodulator octopamine plays an important role in regulating flight speed, and propose a neural circuit for how this controller might be implemented in the flies brain. Two general biological and engineering principles are evident across my experiments: (1) complex behaviors, such as foraging, can emerge from the interactions of simple independent sensory-motor modules; (2) flies control their behavior in such a way that simplifies complex estimation problems.

Short Homologous Sequences Are Strongly Associated with the Generation of Chimeric RNAs in Eukaryotes

Chimeric RNAs have been reported in varieties of organisms and are conventionally thought to be produced by trans-splicing of two or more distinct transcripts. Here, we conducted a large-scale search for chimeric RNAs in the budding yeast, fruit fly, mous

转座子Minos介导的果蝇转基因平台构建及水稻高表达启动子的克隆 任娟 指

本实验室果蝇研究工作，主要集中在黑腹果蝇的新基因起源的研究。新基因起源的分子机制主要包括：外显子重排、基因复制、基因逆转座、移动元件介导、基因水平转移、基因从头起源、基因的断裂融合。为了阐述这些新基因的产生和它们所带来的物种适应性，我们对这些新近起源的基因进行了功能研究。但是，仅仅限于新基因所在物种的功能研究并不能完全解释新基因产生的进化原因，我们需要了解它是否能够给没有该基因的果蝇物种带来一定的适应性。例如一些生殖相关新基因，如果我们将它们转入没有该基因的果蝇，那是否能够给该果蝇带来生殖能力的提高？无论结果如何，这都为我们研究新基因的起源提供一个重要线索。由此，黑腹果蝇以外的其它果蝇物种中实现转基因成为该研究的重要技术环节。但是，实验室目前的转基因系统仅限于P转座子介导的黑腹果蝇转基因系统，因而我们需要建立一种新的转基因平台。而转座子Minos打破物种范围的转基因特性，以及它的转座特点为我们提供了选择。转座子Minos是从果蝇D. hydei中克隆出来长约1.8kb的Ⅱ型转座子，Tc1家族转座元件成员。Minos的转座机制与大部分转座子一样，在宿主基因组里面实行着剪切和粘贴的运作机制。Minos在转座时，偏向插入TA位点并且主要集中于内含子区域，这样可以减少对插入位置基因的影响。此外，Minos在黑腹果蝇中的转座效率约30%，并且拥有一套成熟的选择标记。因此，Minos成为我们解决非黑腹果蝇转基因技术难题的首选。 在本文的工作中，我们采用由希腊Savakis教授（希腊分子生物学与生物技术研究所）提供的Minos转基因系统，完成果蝇的转基因实验。在这套转基因系统中，非自主的转座子Minos和转座酶基因被克隆到了不同载体当中。其中Minos转座子序列中插入了由3xP3眼睛特异表达的启动子介导表达的eGFP报告基因，而转座酶基因则由热激蛋白hsp70启动子调控表达。实验过程中，我们在果蝇D. melanogaster 和D. yakuba的胚胎中分别同时显微注射入含有转座子和转座酶本实验室果蝇研究工作，主要集中在黑腹果蝇的新基因起源的研究。新基因起源的分子机制主要包括：外显子重排、基因复制、基因逆转座、移动元件介导、基因水平转移、基因从头起源、基因的断裂融合。为了阐述这些新基因的产生和它们所带来的物种适应性，我们对这些新近起源的基因进行了功能研究。但是，仅仅限于新基因所在物种的功能研究并不能完全解释新基因产生的进化原因，我们需要了解它是否能够给没有该基因的果蝇物种带来一定的适应性。例如一些生殖相关新基因，如果我们将它们转入没有该基因的果蝇，那是否能够给该果蝇带来生殖能力的提高？无论结果如何，这都为我们研究新基因的起源提供一个重要线索。由此，黑腹果蝇以外的其它果蝇物种中实现转基因成为该研究的重要技术环节。但是，实验室目前的转基因系统仅限于P转座子介导的黑腹果蝇转基因系统，因而我们需要建立一种新的转基因平台。而转座子Minos打破物种范围的转基因特性，以及它的转座特点为我们提供了选择。转座子Minos是从果蝇D. hydei中克隆出来长约1.8kb的Ⅱ型转座子，Tc1家族转座元件成员。Minos的转座机制与大部分转座子一样，在宿主基因组里面实行着剪切和粘贴的运作机制。Minos在转座时，偏向插入TA位点并且主要集中于内含子区域，这样可以减少对插入位置基因的影响。此外，Minos在黑腹果蝇中的转座效率约30%，并且拥有一套成熟的选择标记。因此，Minos成为我们解决非黑腹果蝇转基因技术难题的首选。 在本文的工作中，我们采用由希腊Savakis教授（希腊分子生物学与生物技术研究所）提供的Minos转基因系统，完成果蝇的转基因实验。在这套转基因系统中，非自主的转座子Minos和转座酶基因被克隆到了不同载体当中。其中Minos转座子序列中插入了由3xP3眼睛特异表达的启动子介导表达的eGFP报告基因，而转座酶基因则由热激蛋白hsp70启动子调控表达。实验过程中，我们在果蝇D. melanogaster 和D. yakuba的胚胎中分别同时显微注射入含有转座子和转座酶所在的质粒。转座酶在37度条件诱导下进行表达，协助Minos完成转座过程。在转基因果蝇的阳性筛选中，我们利用眼睛特异表达的绿色荧光蛋作为选择标记。并且，我们通过PCR实验进一步验证了转基因果蝇的真实性。本研究中，我们对转基因实验条件进行了初步优化。我们通过对黑腹果蝇白眼突变品系W1118和D. yakuba注射后胚胎进行保湿，对D. yakuba注射胚胎进行非退壳处理。在改进条件下W1118和D. yakuba的存活率分别为10%和3%左右。通过筛选转基因阳性果蝇，我们得出Minos在W1118和D. yakuba中的转座效率分别在32%和20%左右。我们的实验结果再一次证实了Minos在果蝇D. melanogaster中可行性。同时，该工作也初步完成了在果蝇D. yakuba 中的第一次Minos介导的转基因实验，为新基因的跨物种功能研究奠定了实验基础。在未来的工作计划中，我们将采用Minos转基因系统，把实验室目前研究的黑腹果蝇新基因导入其它物种果蝇进行功能研究。 水稻是一种重要的世界粮食作物，世界上过半的人口以水稻为主食。水稻相对别的粮食作物来讲具有较小的基因组，并且拥有较好的基因组注释，是一种理想的单子叶模式生物。植物转基因技术的发展推动着水稻功能基因组学的研究，目前水稻的转基因技术主要依赖于土壤细菌农杆菌（Agrobacterium tumefaciens）T-DNA介导的外源基因染色体插入。在自然状态下，农杆菌的T-DNA位于Ti致瘤质粒当中。它包括了一些转座元件和一些帮助T-DNA转座的毒性蛋白基因和调节基因。由于Ti质粒上的T-DNA太长，并且没有太多的酶切位点，因此自然状态的T-DNA不适合进行转基因实验。为了方便T-DNA的实际应用，研究人员创立了双载体转基因系统。T-DNA转座区被分离到出Ti载体，并且装载到另外一个适合实验操作的质粒当中，而毒性蛋白表达基因等则保留在Ti质粒上。因此，在进行T-DNA介导的转基因实验时，需要同时存在T-DNA载体和Ti质粒。 本文以“水稻注释计划数据库RAP-DB”的表达数据为参考，选择了60个高表达基因的启动子区域进行克隆。通过对T-DNA载体pCAMBIA1301 进行改造，去掉其原来的35S启动子，将预测的基因启动子克隆到该载体中并与报告基 摘要 因GUS 基因融合。通过分子克隆实验，我们得到了45个高表达基因的启动子载体。最终，为了测试这45个启动子的启动效率，我们会将它们转化到水稻愈伤组织中通过启动子融合的GUS基于表达情况来判断我们启动子的启动效率。

The cDNA cloning and mRNA expression of a potential selenium-binding protein gene in the scallop Chlamys farreri

Selenium binding proteins (SeBP) represent a family of proteins that are believed to be involved in controlling the oxidation/reduction in many physiological processes. The cDNA of Zhikong Scallop Chlamys farreri selenium binding protein (zSeBP) was cloned by expressed sequence tag (EST) and RACE techniques. The high similarity of zSeBP deduced amino acid sequence with the SeBP in other organisms, such as bird, fish, frog, mosquito, fruit fly, mammalian, and even nematode and microorganism indicated that zSeBP should be a member of SeBP family. The temporal expression of zSeBP in the hemocytes was measured by semi-quantitative RT-PCR after scallops were stimulated by either oxidative stress or microbial challenge. The expression of zSeBP was up-regulated progressively after stimulation, and then dropped gradually to the original level. Meanwhile, malondialdehyde (MDA) measured by the colorimetric method in the microbial challenged scallops increased immediately after scallops was challenged by microbes, and was significantly higher than that in the control scallops. Results indicated that the microbial infection could incense the disorder of oxidation/reduction and may result in high MDA production. The negative correlation between the expression level of zSeBP and the MDA content suggested that zSeBP could play an important role in mediating the anti-oxidation mechanisms and immune response in marine invertebrates. (c) 2005 Published by Elsevier Ltd.

The manganese superoxide dismutase gene in bay scallop Argopecten irradians: Cloning, 3D modelling and mRNA expression

A novel manganese superoxide dismutase (MnSOD) was cloned from bay scallop Argopecten irradians by 3' and 5' rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of MnSOD was of 1207 bp with a 678 bp open reading frame encoding 226 amino acids. The deduced amino acid sequence contained a putative signal peptide of 26 amino acids. Sequence comparison showed that the MnSOD of A. irradians shared high identity with MnSOD in invertebrates and vertebrates, such as MnSOD from abalone Haliotis discus discus (ABG88843) and frog Xenopus laevis (AAQ63483). Furthermore, the 3D structure of bay scallop MnSOD was predicted by SWISS-MODEL Protein Modelling Server and compared with those of other MnSODs. The overall structure of bay scallop MnSOD was similar to those of zebrafish Danio rerio, fruit fly Drosophila melanogaster, Chinese shrimp Fenneropenaeus chinensis, human Homo sapiens, and had the highest similarity to scallop Mizuhopecten yessoensis and abalone H. discus discus. A quantitative real-time PCR (qRT-PCR) assay was developed to detect the mRNA expression of MnSOD in different tissues and the temporal expression in haemocytes following challenge with the bacterium Vibrio anguillarum. A higher-level of mRNA expression of MnSOD was detected in gill and mantle. The expression of MnSOD reached the highest level at 3 h post-injection with V. anguillarum and then slightly recovered from 6 to 48 h. The results indicated that bay scallop MnSOD was a constitutive and inducible protein and thus could play an important role in the immune responses against V anguillarum infection. (c) 2008 Elsevier Ltd. All rights reserved.

Bromuro de metilo como tratamiento cuarentenario para cítricos infestados por la mosca del Mediterráneo

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