Extraordinary pH - the mechanism of generation of pH 12 in living systems

Lead Research Organisation: University of Glasgow
Department Name: College of Medical, Veterinary &Life Sci

Abstract

Insects are both vital and deadly in their interactions with humans. Although nearly all the world's crops are pollinated by insects, over a million people a year die from insect-borne diseases, and 20% of the world's crops are lost to insect attack. Insects are becoming resistant to many of our most useful insecticides, and few new ones have been brought to market in the last decade. An improved understanding of what makes insects so successful (in terms of species, they are the dominant life form on earth!) is thus vital in allowing us to find new, selective ways of eliminating harmful pests. Many of the most harmful insects (biting flies and mosquito vectors of disease, and crop-destroying caterpillars) have a specialization that is unique in biology; they run part of their guts at extraordinarily alkaline pH, as high as 12. If we can find out how this high pH is generated, we have a unique, selective target that allows us to attack harmful insects selectively. Our strategy is to identify genes that are expressed at high levels in the high-pH regions of the guts of the malaria and yellow fever mosquitoes, and the Bombyx silkworm caterpillar. Such genes will then be knocked out, either in these species directly, or in Drosophila (the fruit fly), a related insect for which genetic tools are particularly powerful. If gut pH is knocked down by such intervention, then we will have identified potential target genes for the development of new insecticides.

Technical Summary

Insects destroy around 20% of the world's crops, and are vectors for diseases that kill over a million people every year. Widespread insecticide resistance and a shortage of new insecticides mandates the search for novel targets. The larvae of two of the most destructive Orders (Lepidoptera and Diptera), run their midguts at exceptionally high pH (10-12), far in excess of anything encountered in vertebrates. The project seeks to elucidate the mechanism of this alkalinisation, both for basic science reasons, and because this unique process could be targeted for the development of new, more selective (i.e. 'greener') insecticides. The strategy is to identify genes that are selectively enriched in alkalinizing regions of the alimentary canals of three species of insect representative of these Orders (Bombyx mori, Lepidoptera; Anopheles gambiae, Diptera; and Drosophila melanogaster, Diptera), and to study the impact of their knockdown on the alkalinizing ability of the midgut. The results will not only provide the most detailed insights yet into a critical and extreme transport process, but will allow us to start to develop assays for the future development of insecticides.

Planned Impact

The proposal is framed with impact in mind. Academic: - we will be producing highly trained staff for the academic or industrial market. This extends beyond the staff directly employed on the project, to other members of the lab, including undergraduate project students. - We will be producing valuable, cross-species, rich expression datasets, of broad interest to those engaged in study of insect biology, pest control or comparative physiology - We will be advancing knowledge of ion transport, and of extreme biology Economic: - We will develop a model for the generation of uniquely high pH, and in doing so provide information that could lead to improved and selective control of insect pests. This is critically important at a time when new insecticides are slow coming to market, and insects are becoming resistant to those that already exist. Societal: - If we are successful, we will have taken steps towards genuinely novel insect control techniques. This will result in improved health and wealth worldwide for populations limited by the activities of pest insects. - It will increase our attractiveness for further inward investment from the agrochemical industry, leading to more skilled job creation and training, and further dialogues with key industrial partners. - At a general level, extreme biology captures the public imagination. Press releases such as 'pH 12 Achilles' heel inside key pest species', and 'the quest for greener insecticides', should trigger great (positive) press interest.

Publications

10 25 50
 
Description Our guts -and those of most mammals- are highly acidic. Most living organisms, however, are insects, and these can have regions of acidity, or alkalinity, or both. If the mechanisms by which these are generated differs significantly from those in mammals, they can potentially be targetted by novel, 'greener' and more selective insecticides.

We have shown that low pH in insect guts cannot be generated by the same K+/H+ ATPase we find in human gastric parietal cells, as there are no such genes in the genome. By contrast, we have used transcriptomics and reverse genetics to identify the genes that ARE involved in generating acidity: this work is presently out to review in Current Biology.

By contrast, the high pH in many insect guts (up to 12!) is unlike anything in mammals, so a completely new mechanism must be invoked. Again, we have used the methodology outlined in the grant to identify a surprising player in generating high pH; the transepithelial transport of amino-acids. This work is currently out to review in PNAS.

We committed to putting our sequence data into the public domain as a resource for interested parties. Our regional RNAseq database for the larval Drosophila gut is not in the public domain at http://flyatlas.gla.ac.uk/MidgutAtlas/index.html, and the original reads are online at the European NucleotideArchive (ENA), at: 01-Mar-2016 | PRJEB11865 (ERP013282) | RNA transcription in regions of the midgut of Drosophila melanogaster larvae having different pH

A significant part of the project was to target these novel processes as candidate insect control mechanisms. Our method for visualizing pH distributions in vivo, by feeding larvae a cocktail of pH-sensitive dyes, was developed into a 96-well screen, and tested with compounds known to inhibit some of the genes we had been able to identify in the study. Since then, we have obtained follow-on funds to screen a 3000-compound diverse set, and have 167 hits. We are presently performing a secondary screen with these hits, and are hoping to approach industry with the results.
Exploitation Route The insect midgut is critical for survival, and so detailed transcriptomic information will inform research projects worldwide.
Our screening know-how and lead compounds should be of interest to the Agrichemical industry. We have been in discussion with two of the big 4 agrichemical companies in the USA as to how to take these results forward, and may use them as part of a planned spin-out in the next year.
Sectors Agriculture, Food and Drink,Environment

URL http://flyatlas.gla.ac.uk/MidgutAtlas/index.html
 
Description Our findings from these data have been presented to two of the major Agrichemical companies in the USA, and we are presently in discussion as to how to take these forward.
First Year Of Impact 2017
Sector Agriculture, Food and Drink,Environment
Impact Types Economic

 
Description NIH Centre grant
Amount $400,000 (USD)
Funding ID DK100227 
Organisation National Institutes of Health (NIH) 
Sector Public
Country United States
Start 03/2013 
End 03/2018
 
Title RNAseq midgut database 
Description RNAseq profiles for different pH regions of midgut. COpublished with a MS. 
Type Of Material Database/Collection of data 
Year Produced 2016 
Provided To Others? Yes  
Impact none yet. 
URL http://www.ebi.ac.uk/ena/data/view/PRJEB11865
 
Description Collaboration with BASF 
Organisation BASF
Country Germany 
Sector Private 
PI Contribution Based on our leading position in insect biology and functional genomics, we have had longstanding collaborations with BASF in RTP, North Carolina. We have performed extensive contract research over 5 years, and continued to discuss possibilities for the development of new targets. We have also tested novel compounds from BASF against in-house assays.
Collaborator Contribution BASF have funded extensive periods of contract research with our group, and paid us for consultancy when testing novel compounds. They have also hosted a FLIP award with our group, led by Professor SHireen Davies, giving us access to leaders in several of the key chemistry, mode-of-action and regulatory groups in North Carolina. This has informed our plans to develop a spin-out from our research.
Impact Large gene expression datasets and fly lines developed. Details are confidential.
 
Description Collaboration with Bayer 
Organisation Bayer
Department Bayer CropScience Ltd
Country United Kingdom 
Sector Private 
PI Contribution Our identification of processes involved in the generation of extreme pH in insect guts has the potential to lead to new, greener, more selective insecticides at a time when there is field resistance to all commercial insecticides. This work has led to approaches from industry.
Collaborator Contribution Bayer approached us to discuss our plans to commercialize this research. They duly completed 2-way NDAs with the University of Glasgow, and held a conference call with us in February 2018. They are excited by the possibilities, and plan to discuss further once the merger with BASF is complete.
Impact N/A
Start Year 2017
 
Description Collaboration with the Mayo Institute for Urology, Rochester, MINN 
Organisation Mayo Clinic
Country United States 
Sector Charity/Non Profit 
PI Contribution We have shown that it is possible to model kidney stones in Drosophila. Kidney stones are a common cause of morbidity, affecting 10% of the world's population, and accounting for 250 000 emergency room admissions annually in the USA alone. We bring the Drosophila methodology to the collaboration, which has been reflected in several publications, and two NIH grants.
Collaborator Contribution Our collaborators bring a network of clinical urologists and veterinarians (dogs and other animals can have high incidence of kidney stones too); and access to NIH funding. They also provide detailed biophysical characterisation of transporters that we have implicated in stone formation.
Impact We have published several papers from the collaboration: 1: Landry GM, Hirata T, Anderson JB, Cabrero P, Gallo CJ, Dow JA, Romero MF. Sulfate and thiosulfate inhibit oxalate transport via a dPrestin (Slc26a6)-dependent mechanism in an insect model of calcium oxalate nephrolithiasis. Am J Physiol Renal Physiol. 2016 Jan 15;310(2):F152-9. doi: 10.1152/ajprenal.00406.2015. Epub 2015 Nov 4. PubMed PMID: 26538444; PubMed Central PMCID: PMC4719044. 2: Chintapalli VR, Kato A, Henderson L, Hirata T, Woods DJ, Overend G, Davies SA, Romero MF, Dow JA. Transport proteins NHA1 and NHA2 are essential for survival, but have distinct transport modalities. Proc Natl Acad Sci U S A. 2015 Sep 15;112(37):11720-5. doi: 10.1073/pnas.1508031112. Epub 2015 Aug 31. PubMed PMID: 26324901; PubMed Central PMCID: PMC4577160. 3: Cabrero P, Terhzaz S, Romero MF, Davies SA, Blumenthal EM, Dow JA. Chloride channels in stellate cells are essential for uniquely high secretion rates in neuropeptide-stimulated Drosophila diuresis. Proc Natl Acad Sci U S A. 2014 Sep 30;111(39):14301-6. doi: 10.1073/pnas.1412706111. Epub 2014 Sep 16. PubMed PMID: 25228763; PubMed Central PMCID: PMC4191759. 4: Miller J, Chi T, Kapahi P, Kahn AJ, Kim MS, Hirata T, Romero MF, Dow JA, Stoller ML. Drosophila melanogaster as an emerging translational model of human nephrolithiasis. J Urol. 2013 Nov;190(5):1648-56. doi: 10.1016/j.juro.2013.03.010. Epub 2013 Mar 7. Review. PubMed PMID: 23500641; PubMed Central PMCID: PMC3842186. 5: Hirata T, Cabrero P, Berkholz DS, Bondeson DP, Ritman EL, Thompson JR, Dow JA, Romero MF. In vivo Drosophilia genetic model for calcium oxalate nephrolithiasis. Am J Physiol Renal Physiol. 2012 Dec 1;303(11):F1555-62. doi: 10.1152/ajprenal.00074.2012. Epub 2012 Sep 19. PubMed PMID: 22993075; PubMed Central PMCID: PMC3532482. 6: Hirata T, Czapar A, Brin L, Haritonova A, Bondeson DP, Linser P, Cabrero P, Thompson J, Dow JA, Romero MF. Ion and solute transport by Prestin in Drosophila and Anopheles. J Insect Physiol. 2012 Apr;58(4):563-9. doi: 10.1016/j.jinsphys.2012.01.009. Epub 2012 Jan 30. PubMed PMID: 22321763; PubMed Central PMCID: PMC3482613. 7: Dow JA, Romero MF. Drosophila provides rapid modeling of renal development, function, and disease. Am J Physiol Renal Physiol. 2010 Dec;299(6):F1237-44. doi: 10.1152/ajprenal.00521.2010. Epub 2010 Oct 6. Review. PubMed PMID: 20926630; PubMed Central PMCID: PMC3006309.
Start Year 2009
 
Description Glasgow Science Week 2016 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact A stand at Glasgow University's open day for the Glasgow Science Festival. Supported by multiple members of our group.
Year(s) Of Engagement Activity 2016