Testing Protozoacidal Activity of Ligand-lytic Peptides Against Termite Gut Protozoa in vitro (Protozoa Culture) and in vivo (Microinjection into Termite Hindgut)

We are developing a novel approach to subterranean termite control that would lead to reduced reliance on the use of chemical pesticides. Subterranean termites are dependent on protozoa in the hindguts of workers to efficiently digest wood. Lytic peptides have been shown to kill a variety of protozoan parasites (Mutwiri et al. 2000) and also protozoa in the gut of the Formosan subterranean termite, Coptotermes formosanus (Husseneder and Collier 2009). Lytic peptides are part of the nonspecific immune system of eukaryotes, and destroy the membranes of microorganisms (Leuschner and Hansel 2004). Most lytic peptides are not likely to harm higher eukaryotes, because they do not affect the electrically neutral cholesterol-containing cell membranes of higher eukaryotes (Javadpour et al. 1996). Lytic peptide action can be targeted to specific cell types by the addition of a ligand. For example, Hansel et al. (2007) reported that lytic peptides conjugated with cancer cell membrane receptor ligands could be used to destroy breast cancer cells, while lytic peptides alone or conjugated with non-specific peptides were not effective. Lytic peptides also have been conjugated to human hormones that bind to receptors on tumor cells for targeted destruction of prostate and testicular cancer cells (Leuschner and Hansel 2004).In this article we present techniques used to demonstrate the protozoacidal activity of a lytic peptide (Hecate) coupled to a heptapeptide ligand that binds to the surface membrane of protozoa from the gut of the Formosan subterranean termite. These techniques include extirpation of the gut from termite workers, anaerobic culture of gut protozoa (Pseudotrichonympha grassii, Holomastigotoides hartmanni,Spirotrichonympha leidyi), microscopic confirmation that the ligand marked with a fluorescent dye binds to the termite gut protozoa and other free-living protozoa but not to bacteria or gut tissue. We also demonstrate that the same ligand coupled to a lytic peptide efficiently kills termite gut protozoa in vitro (protozoa culture) and in vivo (microinjection into hindgut of workers), but is less bacteriacidal than the lytic peptide alone. The loss of protozoa leads to the death of the termites in less than two weeks.In the future, we will genetically engineer microorganisms that can survive in the termite hindgut and spread through a termite colony as "Trojan Horses" to express ligand-lytic peptides that would kill the protozoa in the termite gut and subsequently kill the termites in the colony. Ligand-lytic peptides also could be useful for drug development against protozoan parasites.Download video file.^{(107M, mov)}     

The Enterobacterium Trabulsiella odontotermitis Presents Novel Adaptations Related to Its Association with Fungus-Growing Termites

Fungus-growing termites rely on symbiotic microorganisms to help break down plant material and to obtain nutrients. Their fungal cultivar, Termitomyces, is the main plant degrader and food source for the termites, while gut bacteria complement Termitomyces in the degradation of foodstuffs, fixation of nitrogen, and metabolism of amino acids and sugars. Due to the community complexity and because these typically anaerobic bacteria can rarely be cultured, little is known about the physiological capabilities of individual bacterial members of the gut communities and their associations with the termite host. The bacterium Trabulsiella odontotermitis is associated with fungus-growing termites, but this genus is generally understudied, with only two described species. Taking diverse approaches, we obtained a solid phylogenetic placement of T. odontotermitis among the Enterobacteriaceae, investigated the physiology and enzymatic profiles of T. odontotermitis isolates, determined the localization of the bacterium in the termite gut, compared draft genomes of two T. odontotermitis isolates to those of their close relatives, and examined the expression of genes relevant to host colonization and putative symbiont functions. Our findings support the hypothesis that T. odontotermitis is a facultative symbiont mainly located in the paunch compartment of the gut, with possible roles in carbohydrate metabolism and aflatoxin degradation, while displaying adaptations to association with the termite host, such as expressing genes for a type VI secretion system which has been demonstrated to assist bacterial competition, colonization, and survival within hosts.     

Genetically Engineered Yeast Expressing a Lytic Peptide from Bee Venom (Melittin) Kills Symbiotic Protozoa in the Gut of Formosan Subterranean Termites

The Formosan subterranean termite, Coptotermes formosanus Shiraki, is a costly invasive urban pest in warm and humid regions around the world. Feeding workers of the Formosan subterranean termite genetically engineered yeast strains that express synthetic protozoacidal lytic peptides has been shown to kill the cellulose digesting termite gut protozoa, which results in death of the termite colony. In this study, we tested if Melittin, a natural lytic peptide from bee venom, could be delivered into the termite gut via genetically engineered yeast and if the expressed Melittin killed termites via lysis of symbiotic protozoa in the gut of termite workers and/or destruction of the gut tissue itself. Melittin expressing yeast did kill protozoa in the termite gut within 56 days of exposure. The expressed Melittin weakened the gut but did not add a synergistic effect to the protozoacidal action by gut necrosis. While Melittin could be applied for termite control via killing the cellulose-digesting protozoa in the termite gut, it is unlikely to be useful as a standalone product to control insects that do not rely on symbiotic protozoa for survival.     

Genetically engineered termite gut bacteria (Enterobacter cloacae) deliver and spread foreign genes in termite colonies

Indigenous gut bacteria of the Formosan subterranean termite (Coptotermes formosanus Shiraki, Isoptera: Rhinotermitidae) were used as shuttle systems to deliver, express and spread foreign genes in termite colonies. The gut bacterium Enterobacter cloacae was transformed with a recombinant plasmid (pEGFP) containing genes encoding ampicillin resistance and green fluorescent protein (GFP). In laboratory experiments, termite workers and soldiers from three colonies were fed with filter paper inoculated with transformed bacteria. Transformed bacteria were detected in termite guts by growing the entire gut flora under selective conditions and checking the cultures visually for fluorescence. We demonstrated that (1) transformed bacteria were ingested within a few hours and the GFP gene was expressed in the termite gut; (2) transformed bacteria established a persistent population in the termite gut for up to 11 weeks; (3) transformed bacteria were efficiently transferred throughout a laboratory colony, even when the donor (termites initially fed with transformed bacteria) to recipient (not fed) ratio was low; (4) transformed E. cloacae were transferred into soil; however, they did not accumulate over time and the GFP plasmid was not transferred to other soil bacteria. In the future, transgenic bacteria may be used to shuttle detrimental genes into termite colonies for improved pest control.     

Protozoacidal Trojan-Horse: Use of a Ligand-Lytic Peptide for Selective Destruction of Symbiotic Protozoa within Termite Guts

For novel biotechnology-based termite control, we developed a cellulose bait containing freeze-dried genetically engineered yeast which expresses a protozoacidal lytic peptide attached to a protozoa-recognizing ligand. The yeast acts as a ‘Trojan-Horse’ that kills the cellulose-digesting protozoa in the termite gut, which leads to the death of termites, presumably due to inefficient cellulose digestion. The ligand targets the lytic peptide specifically to protozoa, thereby increasing its protozoacidal efficiency while protecting non-target organisms. After ingestion of the bait, the yeast propagates in the termite''s gut and is spread throughout the termite colony via social interactions. This novel paratransgenesis-based strategy could be a good supplement for current termite control using fortified biological control agents in addition to chemical insecticides. Moreover, this ligand-lytic peptide system could be used for drug development to selectively target disease-causing protozoa in humans or other vertebrates.     

Ultrastructural studies of the termite (Odontotermes obesus) gut microflora and its cellulolytic properties

The major gut microflora colonizing the hind gut of a higher termite,Odontotermes obesus, included morphologically diverse bacteria, both coccoid and rod-shaped, along with spirochaetes, pseudomonads and actinomycetes. Flagellated protozoa were totally absent. When the gut extract was inoculated on plates containing carboxymethyl cellulose or cellobiose, higher numbers of bacteria grew than on plates without cellulosic sources. The gut homogenate exhibited strong hydrolytic activity when carboxymethyl cellulose,p-nitrophenyl--d-glucoside or xylan were used as substrate, indicating the role of gut microbiota in the process of cellulose and hemicellulose digestion. Activities were highest in the hind gut, and the paunch was probably the major site of polysaccharide digestion in this higher termite.In vitro cultivation of some of the isolates revealed both cellulase and xylanase activities. To our knowledge, this is the first report on ultrastructural studies of the higher termiteOdontotermes obesus.     

Role of bacteria in maintaining the redox potential in the hindgut of termites and preventing entry of foreign bacteria

The hindgut of the lower termites, Mastotermes darwiniensis and Coptotermes lacteus and the higher termite Nasutitermes exitiosus were made aerobic by exposure of the termites to pure oxygen, a procedure which killed their spirochaetes and their protozoa (lower termites only). The time taken for the hindgut to become anaerobic after the termites were restored to normal atmospheric conditions ranged from 2 to 4.5 hr. After oxygen treatment the number of gut bacteria increased some six- to ten-fold in all termite species, indicating that the bacteria are poised to use oxygen entering the gut. Removal of all the hindgut microbiota by feeding tetracycline caused the hindgut to become aerobic in M. darwiniensis and N. exitiosus. The transferring of M. darwiniensis to fresh wood, free of antibiotic, resulted in the return of the normal flora and the eventual establishment of anaerobic conditions in the hindgut. Thus the bacteria appear to be important in maintaining anaerobic conditions in the gut. Attempts to determine whether the protozoa (in the lower termites) played any part in maintaining the Eh of the hindgut were unsuccessful. Serratia marcescens failed to colonise the gut of normal C. lacteus and transiently colonized (for 5 days) the gut of normal N. exitiosus. Transient colonization by S. marcescens (from 6 to 10 days) occurred in N. exitiosus when its hindgut spirochaetes were killed and in C. lacteus when its spirochaetes and protozoa were killed, indicating a possible role for the spirochaetes and/or protozoa in influencing the bacteria allowed to reside in the hindgut. Exposure of normal termites to Serratia provoked an increase in the numbers of the normal gut bacteria.     

Transformed bacterial symbionts re-introduced to and detected in host gut

Strains of Enterobacter agglomerans and Klebsiella pneumoniae isolated from Rhagoletis completa Cresson were engineered to express transgenic fluorescent proteins (ECFP, DsRed). These bacteria were introduced into flies by feeding the flies a sucrose solution in which the bacteria were suspended. The transgenic and heterologous marker protein was expressed and visible in the bacteria after they were ingested by WHF and while they were in the fly gut. We describe the plasmids used to transform these bacteria and demonstrate expression of heterologous proteins from the transforming plasmids and discuss the implications for future pest control strategies. Received: 14 September 2001 / Accepted: 22 October 2001     

Iron reduction in the metal-rich guts of wood-feeding termites

Termites play important roles in lignocellulose and humus turnover in diverse terrestrial ecosystems, and are significant sources of global atmospheric methane and carbon dioxide. All known termite species engage in obligate, complex nutritional symbioses with their gut microbes to carry out such processes. Several hundred microbial species, representing a broad phylogenetic and physiological diversity, are found within the well‐bounded, microliter‐in‐scale gut ecosystem of a given termite. However, most of these species have never been obtained in laboratory culture, and little can be said about their functional roles in the gut community or symbiosis. Herein, an unappreciated facet of the gut chemistry and microbiology of wood‐feeding termites is revealed: the redox metabolism of iron. Gut fluids from field‐collected termites contained millimolar amounts of ferrous iron and other heavy metals. When iron(III) hydroxides were amended to a filter paper diet of Zootermopsis nevadensis, a dampwood termite collected in the San Gabriel Mountains of Southern California, the specimens accumulated high levels of iron(II) in their guts. Additionally, iron was reduced at rapid initial rates in anoxic gut homogenates prepared from field‐collected Z. nevadensis specimens. A Clostridium sp. and a Desulfovibrio sp. were isolated from dilution‐to‐extinction enrichments of Z. nevadensis gut contents and were found to reduce iron(III), as did the termite gut spirochete Treponema primitia. The iron in the guts of wood‐feeding termites may influence the pathways of carbon‐ and electron‐flow, as well as microbial community composition in these tiny ecosystems of global importance.     

Use of Genetically Engineered Escherichia coli to Monitor Ingestion, Loss, and Transfer of Bacteria in Termites

Escherichia coli was transformed with a recombinant plasmid (pEGFP) containing the genes for ampicillin resistance and Green Fluorescent Protein (GFP). Escherichia coli expressing GFP (E. coli/GFP+) was then fed to workers of the termite Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae). The transformed bacteria in the termite guts were detected by growing the gut flora under selective conditions and then checking the cultures for fluorescence. Recombinant plasmids in the termite gut were detected by plasmid extraction with subsequent restriction enzyme digest. The presence of the GFP gene in the gut of termites fed with E. coli/GFP+ was verified by PCR amplification. Transformed E. coli were ingested rapidly when workers fed on filter paper inoculated with E. coli/GFP+. After 1 day, 42% of termite guts harbored E. coli/GFP+. Transfer of E. coli/GFP+ from donor termites (fed with E. coli/GFP+) to recipients (fed with moist filter paper) occurred within 1 day. However, without continuous inoculation, termites lost the transformed bacteria within 1 week.     

Symbiotic adaptation of bacteria in the gut of Reticulitermes speratus: Low endo-β-1,4-glucanase activity

The termite is a good model of symbiosis between microbes and hosts and possesses an effective cellulose digestive system. Oxygen-tolerant bacteria, such as Dyella sp., Chryseobacterium sp., and Bacillus sp., were isolated from Reticulitermes speratus gut. Notably, the endo-β-1,4-glucanase (EG) activity of all 16 strains of isolated bacteria was low. Due to the combined activity of EG from the termites and their symbiotic protozoa, the bacteria might not be compelled to express EG. This observation demonstrates how well intestinal bacteria have assimilated themselves into the efficient cellulose digestive systems of termites.     

Diversity of Gut Bacteria of <Emphasis Type="Italic">Reticulitermes flavipes</Emphasis> as Examined by 16S rRNA Gene Sequencing and Amplified rDNA Restriction Analysis

The phylogenetic species richness of the bacteria in the gut of the termite Reticulitermes flavipes was examined using near full-length 16S rRNA gene sequencing and amplified rDNA restriction analysis (ARDRA). We amplified the genes by polymerase chain reaction (PCR) directly from a mixed population of termite gut bacteria and isolated them using cloning techniques. Sequence analysis of 42 clones identified a broad taxonomic range of ribotypes from six phyla within the domain Bacteria: Proteobacteria, Spirochaetes, Bacteroidetes, Firmicutes, Actinobacteria, and the recently proposed “Endomicrobia.” Analysis of the sequence data suggested the presence of a termite specific bacterial lineage within Bacteroidetes. The ARDRA data included 261 different ARDRA profiles of 512 clones analyzed. These data suggest the gut flora in R. flavipes is extremely diverse.     

Isolation of Nitrogen Fixing Bacteria from Fungus Termites

Biological nitrogen fixation by the microorganisms in the gut of termites is one of the singularly important symbiotic processes, since termites invariably thrive on nitrogen poor diet. Two isolates of free living aerobic and facultative anaerobic N fixing bacteria were obtained from the guts of fungus cultivating termite, Macrotermes sp. Among the total bacterial isolates from termite gut, the per cents of N fixing aerobes viz., Azotobacter and Beijerinckia spp were 49% and 37% from the salivary gland while facultative N fixing anaerobe viz., Klebsiella and Clostridium contributed (51% and 93%). The free living aerobic bacteria were identified as Azotobacter spp (19 x 10⁴ CFU mL^‐1) and Beijerinckia (13.2 x 10⁴ CFU mL^‐1) from the salivary gland of the termite; interestingly, foregut, mid gut and hind gut registered a low population of these bacteria. The isolates of Azotobacter were smooth, glistening, vicid in nature, rods, gram negative and cyst forming. Isolates of Beijerinckia sp. produced copious slime, tenacious, rods, gram negative with no cyst formations. Both the isolates emitted green fluorescence and produced acid. Facultative N fixing anaerobes were harbored in the hind gut. The isolates were identified as Klebsiella (20 x 10⁴ CFU mL^‐1) and Clostridium pasteurianum 39.1 x 10⁴ CFU mL^‐1. Klebsiella were straight rods arranged singly or in pairs, non‐motile, gram negative, whereas Clostridium pasteurianum was viscoid, motile with terminal spores. A positive correlation was observed between the extractable polysaccharides of these isolates and soil aggregation. The aggregates formed by the isolates increased soil aeration, porosity, water holding capacity and helped in better plant growth. Thus, the gut microflora of termite, apart from harnessing nitrogen from the atmosphere, also helps improving soil fertility.     

Comparison of gut-associated and nest-associated microbial communities of a fungus-growing termite (Odontotermes yunnanensis)

Abstract The digestion of cellulose by fungus-growing termites involves a complex of different organisms, such as the termites themselves, fungi and bacteria. To further investigate the symbiotic relationships of fungus-growing termites, the microbial communities of the termite gut and fungus combs of Odontotermes yunnanensis were examined. The major fungus species was identified as Termitomyces sp. To compare the micro-organism diversity between the digestive tract of termites and fungus combs, four polymerase chain reaction clone libraries were created (two fungus-targeted internal transcribed spacer [ITS]– ribosomal DNA [rDNA] libraries and two bacteria-targeted 16S rDNA libraries), and one library of each type was produced for the host termite gut and the symbiotic fungus comb. Results of the fungal clone libraries revealed that only Termitomyces sp. was detected on the fungus comb; no non-Termitomyces fungi were detected. Meanwhile, the same fungus was also found in the termite gut. The bacterial clone libraries showed higher numbers and greater diversity of bacteria in the termite gut than in the fungus comb. Both bacterial clone libraries from the insect gut included Firmicutes, Bacteroidetes, Proteobacteria, Spirochaetes, Nitrospira, Deferribacteres, and Fibrobacteres, whereas the bacterial clone libraries from the fungal comb only contained Firmicutes, Bacteroidetes, Proteobacteria, and Acidobacteris.     

Cloning and Heterologous Expression of Insecticidal-Protein-Encoding Genes from Photorhabdus luminescens TT01 in Enterobacter cloacae for Termite Control

Enterobacter cloacae, one of the indigenous gut bacteria of the Formosan subterranean termite (Coptotermes formosanus), was genetically modified with a transposon Tn5 vector containing genes (tcdA1 and tcdB1) encoding orally insecticidal proteins from the entomopathogenic bacterium Photorhabdus luminescens subsp. laumondii TT01, a symbiont of the entomopathogenic nematode Heterorhabditis bacteriophora, for termite control. In the laboratory, termites were fed filter paper inoculated with the recombinant bacteria. The chromosomal expression of the introduced genes showed that there were insecticidal activities against termite workers and soldiers challenged with the transformed bacteria. After termites were fed recombinant bacteria, the termite mortality was 3.3% at day 5, and it increased from 8.7% at day 9 to 93.3% at day 29. All the dead termites contained the recombinant bacteria in their guts. Transfer of the recombinant bacteria occurred between donor workers (initially fed recombinant bacteria) and recipient workers (not fed). More than 20% of the recipient termites ingested recombinant bacteria within 2 h, and 73.3% of them had ingested recombinant bacteria after 12 h. The method described here provides a useful alternative for sustainable control of the Formosan subterranean termite (C. formosanus) and other social insects, such as the imported red fire ant (Solenopsis invicta).     

Association of methanogenic bacteria with flagellated protozoa from a termite hindgut

Epifluorescence microscopy was used to examine hindgut contents ofZootermopsis angusticollis (Hagen) termites for the presence of methanogenic bacteria, which can be identified on the basis of the fluorescence of the novel cofactors F₄₂₀ and F₃₅₀. Small, autofluorescent, rod-shaped bacteria of theMethanobrevibacter sp. morphotype were observed associated with three flagellates tentatively identified asTrichomitopsis termopsidis (Cleveland),Tricercomitus termopsidis Kirby andHexamastix termopsidis Kirby. Methanogens were not observed associated with any other protozoal morphotypes and were not numerous in the free-living state inZ. angusticollis hindgut fluid. Electron micrographs of thin sections of hindgut protozoa suggest methanogens are endosymbionts in the small trichomonad protozoa. Our observations are consistent with the finding of Odelson and Breznak that methane is a minor endproduct of the metabolism of termite gut microbiota.Deceased.     

The hindgut lumen prokaryotic microbiota of the termite Reticulitermes flavipes and its responses to dietary lignocellulose composition

Reticulitermes flavipes (Isoptera: Rhinotermitidae) is a highly eusocial insect that thrives on recalcitrant lignocellulosic diets through nutritional symbioses with gut‐dwelling prokaryotes and eukaryotes. In the R. flavipes hindgut, there are up to 12 eukaryotic protozoan symbionts; the number of prokaryotic symbionts has been estimated in the hundreds. Despite its biological relevance, this diverse community, to date, has been investigated only by culture‐ and cloning‐dependent methods. Moreover, it is unclear how termite gut microbiomes respond to diet changes and what roles they play in lignocellulose digestion. This study utilized high‐throughput 454 pyrosequencing of 16S V5‐V6 amplicons to sample the hindgut lumen prokaryotic microbiota of R. flavipes and to examine compositional changes in response to lignin‐rich and lignin‐poor cellulose diets after a 7‐day feeding period. Of the ~475 000 high‐quality reads that were obtained, 99.9% were annotated as bacteria and 0.11% as archaea. Major bacterial phyla included Spirochaetes (24.9%), Elusimicrobia (19.8%), Firmicutes (17.8%), Bacteroidetes (14.1%), Proteobacteria (11.4%), Fibrobacteres (5.8%), Verrucomicrobia (2.0%), Actinobacteria (1.4%) and Tenericutes (1.3%). The R. flavipes hindgut lumen prokaryotic microbiota was found to contain over 4761 species‐level phylotypes. However, diet‐dependent shifts were not statistically significant or uniform across colonies, suggesting significant environmental and/or host genetic impacts on colony‐level microbiome composition. These results provide insights into termite gut microbiome diversity and suggest that (i) the prokaryotic gut microbiota is much more complex than previously estimated, and (ii) environment, founding reproductive pair effects and/or host genetics influence microbiome composition.     

Gut microflora of two saltmarsh detritivore thalassinid prawns,Upogebia africana andCallianassa kraussi

The presence and digestive capabilities of bacteria associated with the digestive systems and habitats of two saltmarsh-burrowing detritivore thalassinid prawns (Upogebia africana andCallianassa kraussi) was examined.U. africana is a filter-feeding prawn inhabiting muddy deposits, whereasC. kraussi, a deposit feeder, inhabits coarser more sandy deposits. Scanning electron microscopy was used to examine the gut lining and associated microflora and the nature of the ingested food of both prawns. The gut contents of both prawns included plant fragments, fragmented diatoms, partially degraded protozoa, and bacteria attached to organic matter. In bothU. africana andC. kraussi the midgut walls and gut contents were extensively coated by filamentous bacteria which were absent in the hindgut. The hindgut epithelium ofU. africana was coated by mats of rodshaped bacteria, not reported in marine invertebrates previously. The digestive glands of both species contained bacteria in the lumen. Isolation of gut and habitat bacteria suggests that bothU. africana andC. kraussi maintain a gut microflora distinct from the habitat microflora. Bacteria isolated from the guts of both species of prawn differed from those isolated from their respective habitats with regards to both the genera isolated and their digestive capabilities. The dominant genera isolated from the guts of bothU. africana andC. kraussi wereVibrio andPseudomonas, with an unidentified fermenter andPseudomonas, respectively dominating in the digestive glands. Bacteria of the genusAcinetobacter dominated the isolates from the habitats of both species of prawn. Resident gut bacteria isolated from the guts of both species of prawn exhibited lipase, protease, chitinase, and lysozyme, but not cellulase activity, and may contribute to nitrogen aquisition by the prawns. Isolates from the prawns' habitat exhibited alginase, gelatinase, and lipase activity, a few (3%) fromU. africana habitat having cellulases. In this study a distinction between resident gut bacteria and transient gut bacteria was made. Results suggest that some habitat bacteria remain viable in the guts ofU. africana, but not inC. kraussi.     

Interspecific Interactions Between Coptotermes formosanus and Reticulitermes flavipes (Isoptera: Rhinotermitidae) in Laboratory Bioassays

Experiments were conducted to examine competitive interactions between the Formosan subterranean termite, Coptotermes formosanus Shiraki (FST), and the eastern subterranean termite, Reticulitermes flavipes (Kollar) (EST), using groups of termites with different worker:soldier proportions. Experiments were conducted using three connected test chambers: an FST chamber, an unoccupied center chamber, and an EST chamber. When groups of FST were comprised of 20% soldiers versus 2% EST soldiers, only 8% of center chambers were occupied exclusively by EST. When groups of FST were comprised of 10% soldiers versus 1% EST soldiers, 44% of center chambers were occupied exclusively by EST. When the only food source was located in the center chamber, 60% of center chambers were occupied by both species. FST did not completely displace EST in any of these experiments.