Location: Pest Management and Biocontrol Research2013 Annual Report
1a. Objectives (from AD-416):
The whitefly Bemisia tabaci is one of the most important pests of agricultural and ornamental crops worldwide, causing direct feeding damage, negatively affecting post-harvest of numerous crops, and transmitting devastating plant viral diseases. B. tabaci feeds exclusively on plant sap, and therefore has evolved a unique digestive system that handles excess dietary fluid and maintains osmotic pressure. Previously, we characterized an aquaporin protein from B. tabaci (BtAQP1) that functions as a water channel protein to transport water through the specialized digestive tract. The objective of the proposed cooperative research is to test novel antagonists specifically developed to target aquaporins and ascertain if these agents interfere with water permeability of the BtAQP1 and thus negatively impact B. tabaci. Compounds optimized for efficacy and specificity against insect aquaporin targets represent a novel insecticidal approach for insect pest control.
1b. Approach (from AD-416):
We propose to first evaluate agents that are fast unidirectional blockers of water flux as well as slower bidirectional blockers against BtAQP1 produced using a frog oocyte expression system. Any compounds found to be effective on the BtAQP1 in the oocyte expression system, would then be tested on the developing and adult whiteflies. Compounds will be tested against B. tabaci by direct application and through oral feeding, using both plants and artificial feeding systems. For promising compounds, we will develop a chemical library of analogs around the active structure, and improve potency for selective channel block and whitefly efficacy.
3. Progress Report:
This Non Funded Cooperative Agreement directly addresses Objective 1 of the inhouse project, "Develop knowledge and control tactics based on the physiology, biochemistry, genetics and vector-pathogen interactions of insect pests", and Subobjective 1.3, "Characterization of gene-silencing of a functional aquaporin water transport protein from B. tabaci". Hemipteran insect pests, including whiteflies and plant bugs, are some of the most destructive and difficult to control insect pests worldwide. They threaten food, fiber, and ornamental crops by direct feeding damage, pathogen transmission, and indirect plant interactions. The plant bug (Lygus hesperus) and the whitefly (Bemisia tabaci) are serious pests of cotton, fruits, vegetables, alfalfa and many other U.S. crops, with control strategies often heavily reliant on broad-spectrum insecticides. These sap-feeding pests use piercing-sucking mouthparts to ingest large quantities of plant fluid and have highly evolved physiological and biochemical mechanisms to process high volume liquid diets and regulate osmotic balance. We hypothesize that disruption of these critical water balance processes may impede homeostasis and impose costs that manipulate pest populations. Aquaporins are membrane channels that can facilitate osmotic water, glycerol and/or other small molecule fluxes across biological membranes in cells. Insects use aquaporin proteins to facilitate movement of water and other small solutes in critical functions such as digestion, excretion, and thermal stress. In B. tabaci, at least one aquaporin (BtAQP1) is located in the specialized gut tract and functions to transport water across tissues for maintenance of osmotic pressure and/or excretion of excess dietary fluid. Because initial attempts to silence BtAQP1 by RNA interference (RNAi) did not demonstrate visible negative effects on whitefly nymphs or adults, it is possible that additional aquaporin proteins are present and provide compensatory water flux. We used transcriptome sequencing from L. hesperus and B. tabaci, as well as data mining from other publically available whitefly sequence databases, to obtain partial reads for six new aquaporin gene-coding sequences from B. tabaci and five from L. hesperus. Molecular cloning techniques were used to obtain full-length DNA sequences that produce proteins strongly resembling aquaporins from other insects. Laboratory tests verified that all five lygus aquaporins and seven whitefly aquaporins were correctly assembled and exist in insect populations. Molecular characterization of lygus aquaporins has shown that some exist in variable forms (that is, some lygus aquaporin proteins are made from the same gene but differ slightly in their composition). While most of the lygus aquaporins are produced throughout developmental stages, some exhibit differential patterns of expression in tissues. Those aquaporins expressed in gut tissue are of key interest, as they are most readily available to target by ingested materials. Furthermore, surrogate host cells (unfertilized frog oocytes) were used to produce all five lygus aquaporin proteins where they demonstrated strong water transport functionality and no transport of glycerol. The oocyte expression system is established within the collaborator’s laboratory at the University of Adelaide and is currently being used to screen novel compounds and/or plant extracts containing putative aquaporin antagonists or protagonists possessing specificity for targeting water channel function. Anecdotal observations indicate that some hemipterans (such as Lygus spp.) are averse to some plants from the Asteraceae family and thus, form the basis for the hypothesis that a compound or compounds from plants found in this family may negatively regulate aquaporins, causing misregulation of water balance in the alimentary tract. When tested in laboratory and greenhouse no-choice experiments, neither whiteflies nor lygus bugs performed well on plants of a selected species of Asteraceae. Preliminary experiments indicate that aqueous and organic extracted fractions from this plant species exhibited different influences on water channel movement against the one lygus aquaporin tested in oocytes. Whereas an aqueous-soluble extract showed strong dose-dependent increase in water channel activity, an organic solvent extract resulted in strong inhibition of water transport. The aqueous-soluble fraction also showed dose-dependent insecticidal activity when fed to adults of both pest species. Further experiments to separate, characterize, and identify the active components of the water-soluble fraction are needed.