David A. O'Brochta - US grants

9407948 Abstract A circadian system was described which controls sperm release and maturation in the make reproductive tract of the gypsy moth. The system was characterized at both the chrono-biological and physiological level, including a time-table for its development during adult metamorphosis. Study of this system at the molecular level was initiated and resulted in the isolation of three clock related(cr) genes whose transcript abundance appear to fluctuate in a circadian fashion. A determination will be made as to whether and how CR genes are involved in the clock mechanism. Changes in the abundance of the three CR genes will be examined during the course of development of circadian system after resetting of the circadian mechanism by light or temperature, and in conditions which disrupt rhythmicity using an RNAse protection assay and reverse transcription-PCR. In addition, cellular localization of mRNAs corresponding to CR probes will be studied by in situ hybridizations. %%% Circadian rhythms are ubiquitous in living organisms at every level or organization, from gene transcription to behavior. Despite the fundamental importance of circadian rhythms, the molecular identity of the clock that drives them remains one of the significant unanswered questions of modern biology. In this study, a simple circadian system of an insect, sperm release and maturation in the male reproductive tract of the gypsy moth, is used to explore the molecular basis of the biological clock. ***

biomedical equipment purchase;

Aedes; Plasmodium; salivary glands; communicable disease transmission; communicable disease control; arthropod borne communicable disease; host organism interaction; epizootiology; transfection; gene expression; arthropod nonpollutant control; malaria; genetic manipulation;

Insect vector-born diseases continue to be a major source of mortality worldwide. The continuous development of new tools to study and combat these diseases and the insects that transmit them is essential. The long term goals of this research program are to develop genetic engineering tools for insects of medical significance to man and to use these new tools to gain a better understanding of the basic biology of insect/pathogen and parasite interactions. These new tools will also be used to develop new genetics-based methods for interfering with insect vector-born disease transmission. This proposal has two principal components - continued development of the Hermes gene vector system to improve its efficiency and host range, and the development of genetic tools to investigate Culex mosquitoes. The specific aims are to 1) understand the modes of integration used by Hermesin mosquito and how these modes are regulated 2) determine the mechanisms, range and consequences of hAT element interactions in insects, 3) increase the recombination activity of Hermes transposase through protein modification and engineering, 4) develop gene vector and enhancer-trapping tools for Culex mosquitoes. 5) develop a functional anopheline hAT element-based gene vector system. These aims will be accomplished using genetic and molecular genetic methods.

DESCRIPTION (provided by applicant): One of the more vexing problems associated with the study of human diseases transmitted by insects over the last twenty years has been the development of genetic transformation technology for the human malaria vector, Anopheles gambiae. Genetic transformation systems for An. gambiae and other insects will be used to study insect/parasite interactions and to develop novel genetics-based strategies for controlling disease transmission. Four insect transposable element-based gene vectors currently exist but none have been successfully used to create transgenic An. gambiae and none are very efficient in those mosquito species in which they have been shown to function. The Tn5 transposable element system from bacteria may provide the key to successfully transforming An. gambiae because it has properties distinctly different from existing insect transposable element-based systems. First, the Tn5 system is biochemically well characterized and has been modified to have 1000 times the activity of the native system. Second, an active intermediate called a synaptic complex or Transposome TM can be produced in vitro and can be introduced directly into the cells to be transformed. Introducing this active intermediate further increases the efficiency of transformation. The use of a hyperactive recombination system and the ability to produce active intermediates in vitro may be an important key to the development of a transformation system for An. gambiae that will have real utility in the laboratory. Preliminary data show that Tn5 is functional in insect germ cells. Here the Tn5 system will be tested in An. gambiae in conjunction with a new protocol for introducing DNA into developing mosquito embryos. Two decades of failed efforts to create transgenic An. gambiae make this is a high risk project but one whose impact on the field of medical entomology and efforts to control the transmission of malaria will be enormous if it is successful.

DESCRIPTION (provided by applicant): Insect-born diseases such as malaria can be controlled in a variety of ways ranging from the treatment of sick patients to eliminating the insects that transmit the pathogens or parasites. The work described here will advance efforts to evaluate the feasibility of applying certain insect biotechnological approaches to the control of malaria transmission. Insect genetics-based strategies for controlling diseases such as malaria in Africa are being developed with some success in the laboratory. Using transgenic insect technologies Anopheles mosquitoes have been created that express a variety of effector-genes that reduce or eliminate the capacity of these insects to support Plasmodium development. While the insects produced to date have not had optimal phenotypes they have served to demonstrate that Plasmodium development and transmission in Anopheles mosquitoes can be disrupted using existing insect genetic engineering technologies. However, in order for effective laboratory-created genotypes to be of any practical use in controlling malaria transmission in natural environments they will have to be introduced into and spread through natural populations of the target species. Technologies for accomplishing this objective have yet to be identified although a number of candidates exist. Class II transposable elements have been suggested as gene spreading agents based on their natural history. Whether any of the existing insect gene vectors could serve to spread anti-Plasmodium transgenes through populations of Anopheles gambiae remains untested. This major deficiency in the efforts to explore the feasibility of the idea of manipulating vector competence for the purposes of disease transmission control will be addressed in the work proposed here. Following the introduction of conditionally autonomous Hermes, Minos, Mos1 and piggyBac elements into An. gambiae their remobilization, replication and spreading potential will be quantitatively assessed. The specific aims are to 1) assess the remobilization potential of Hermes, Minos, Mos1 and piggyBac in An. gambiae, 2) determine the patterns of remobilization of Hermes, Minos, Mos1 and piggyBac in An. gambiae, 3) assess the replicative potential of Hermes, Minos, Mos1 and piggyBac in An. gambiae, 4) assess the spreading potential of Hermes, Minos, Mos1 and piggyBac in An. gambiae. At the end of this project the relative promise of each of these transposable elements to contribute to the development of population replacement technology for An. gambiae will be known. This project involves the use of Animals but does not involve Human Subjects.

DESCRIPTION (provided by applicant): Insect vector-borne diseases continue to burden a large fraction of the world's population. Understanding the basic biology of the insects responsible for transmitting the pathogens that cause these diseases has been and will continue to be essential for the development of effective strategies of control. Technological advances often provide novel opportunities to enlarge our understanding of basic biology. This project will result in the creation of a genetic resource consisting of transgenic lines of the human malaria mosquito Anopheles stephensi expressing the transcription factor Gal4 specifically in hemocytes. This collection of lines will serve as a valuable tool for the investigation of the cellular immune system in mosquitoes, which consists of hemocytes that are responsible for encapsulation and phagocytosis as well as synthesis of antimicrobial proteins. This aspect of the mosquito's immune system is less well understood than the more-well-studied humoral immune system although there has been growing realization that it is an important mediator of microbial infections in these insects. This collection of lines expressing Gal4 specifically in hemocytes will be used subsequently to label hemocytes in vivo to facilitate their visualization under a variety of physiological conditions, to manipulate hemocyte gene expression (silencing, overexpression, mis-expression) to assess the genetic basis of hemocyte growth, development and/or function and to express cell-death or cytotoxic genes to specifically ablate hemocytes or subpopulations of hemocytes to assess the impact on various physiological processes.

DESCRIPTION (provided by applicant): Insect vector-borne diseases continue to burden a large fraction of the world's population. Understanding the basic biology of the insects responsible for transmitting the pathogens that cause these diseases has been and will continue to be essential for the development of effective strategies of control. Technological advances often provide novel opportunities to enlarge our understanding of basic biology. This project will result in the creation of enhancer- and gene-trap technologies for the human malaria mosquito Anopheles stephensi that will enable researchers to identify, isolate and analyze genes in new and powerful ways. The technology will be applied to the study of the salivary glands, an organ that plays a vital role in the transmission of pathogens such as Plasmodium. Preliminary results show that the transposable element piggyBac is highly active within the genome of Anopheles stephensi, making it a useful platform upon which to build these genetic technologies. Prototype enhancer- trap and gene-trap systems have been created and introduced into Anopheles stephensi. Preliminary screens to test the functionality of both systems were successfully completed. Both systems are active and effective at sensing enhancers and genes, respectively. A small diverse collection of enhancer-trap lines is described which provides a proof of principle. Based on prototype designs, existing Anopheles stephensi enhancer- and gene-trap technologies will be modified for increased efficiency and effectiveness. The transgenic lines necessary for conducting enhancer- and gene-trap screens, as well as, useful 'Gal4 driver lines' will become a community resources. Anopheles stephensi gene-trap technology will be used to identify and isolate genes specifically expressed in the salivary glands of larvae and adults under a variety of developmental and physiological conditions. This project will enable the importance of various salivary gland secretions in mosquito feeding and pathogen transmission to be understood, and to integrate knowledge concerning salivary gland structure and function. This new detailed understanding of this critical tissue in a major vector o human disease is expected to lead to the conception and implementation of new strategies for disrupting pathogen transmission.

Insects are second to no animal in their importance to humans; consider their roles in pollination, devouring crops and transmitting diseases. Our success in living with or without insects depends upon the depth of our understanding of insect biology. Molecular and genome sciences are revolutionizing our understanding of human biology and medicine, and it can do the same for the study of insects. There is a major problem; developing advanced molecular and genetic technologies for one species, humans, has been challenging but developing similar technologies for hundreds of important insect species currently studied intensely by scientists is immensely more challenging. For some insects the technologies are well developed and knowledge of their application is deep (fruitflies), while for other insects the technologies are nonexistent and technical knowledge is shallow (honeybees). This NSF-Research Coordination Network fosters the development and sharing of molecular and genetic knowledge and technologies across diverse research groups. The Network fosters cross-disciplinary interactions by convening training workshops and classes, sponsoring symposia, promoting collaborations and assembling an online knowledge emporium for insect scientists.

This project will have four major impacts: 1) enabling solutions to society's most vexing insect-related problems, e.g. overuse of insecticides and reemergence of insect-borne diseases, 2) maximizing the value of research-dollars by encouraging collaborations and leveraging existing resources, 3) preparing for the future by training students, 4) enhancing participation of underrepresented groups in science.