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Evaluation of the effect of zwitterionic buffers on termite gut protists
2018 - IRG/WP 18-10933
The thermal modification of wood is recognized as the most commercialized wood modification process. The treatment of wood at temperatures usually greater than 180 ºC results in a product with some properties enhanced (dimensional stability, durability, aesthetical colour) and some diminished (mechanical properties). The loss of mechanical strength is recognized as a limitation to the use of thermally modified wood in certain products. The mechanical strength reduction is linked in varying degrees to the release of acidic volatile species, the acid-catalysed depolymerisation of the hemicelluloses present and the plasticization and redistribution of the lignin components present. Buffers, although been designed not to influence the reaction systems, may have some interactions when exposed to some conditions, potentially acting as promoters of biological changes on different systems. In this study, two zwitterionic buffers, bicine and tricine, were chosen to be tested regarding their volatilization reduction properties, as well as tricine due to offering the potential of forming Maillard-type products with fragmented hemicelluloses/volatiles. In order to determine if there were any resultant effects of the bicine and tricine treatments submitted or not to thermal modification, the efficacy against subterranean termites was conducted as well as an evaluation of the effect of the different combinations of treatments on the termite gut symbiotic protists. The durability of thermal modified wood is recognized as low and the results of the present work are in accordance with that perception. However, bicine and tricine treatments alone had a clear influence on the survival of the termites and the study of the protist symbionts (9 morphotypes) gave a better insight of the ability of termites to react to unfavorable diet changes.
S Duarte, D Jones, L Nunes


Effect of Rifampin on gut symbiotic bacteria isolated from Anacanthotermes vagans Hagen (Isoptera: Hodotermitidae)
2014 - IRG 14-10835
Termites thrive in terrestrial ecosystems and play an important role in bio-recycling of the lignocellulosic biomass, that is a mixture of cellulose, hemicellulose and lignin. However, these insects become pests when they interfere with human interests related to wood/cellulose products, attacking structural timber, furniture and paper products. Sustainable management of subterranean termites using biorational pesticides such as antibiotics could represent a promising alternative to chemical control. We assessed the efficacy of the antibiotic Rifampin against the symbiotic bacteria isolated from the termite Anacanthotermes vagans using qualitative methods. Rifampin was dissolved in Dimethylsulfoxide (DMSO). We used a broth-dilution technique for determining Rifampin minimum inhibitory concentration (MIC) on isolated bacteria from gut of A.vagans maintained at 37ᵒC for 24h in an incubator. Summarizing the results, among the isolated species, Enterobacter cloacae, Rhodococcus sp. and Bacillus subtilis were sensitive to the action of this antibiotic, whereas Klebsiella pneumonia, Enterobacter aerogenes, Enterobacter sp., Klebsiella oxytoca, Klebsiella sp., Corynebacterium sp., and Staphylococcus lentus were less affected by this antibiotic.
B Habibpour, M Jalali, D Gharibi


Effects of geographical and dietary variation on the symbiotic flagellate protists communities of the subterranean termite Reticulitermes grassei Clément
2015 - IRG/WP 15-10847
Despite their importance on diverse ecosystems, termites may also be considered severe pests of wood in service, and also as agricultural and forestry pests. Subterranean termites’ ability to digest lignocellulose relies not only on their digestive tract physiology, but also on the symbiotic relationships established with flagellate protists and bacteria. In this tripartite lignocellulolytic system, the termite contribute with endogenous cellulases and mechanical processing, flagellate protists phagocyte the wood particles and digest them, and prokaryotes have, among others, an important role in maintaining the physical-chemical equilibrium inside the termite hindgut. The flagellate protist community living inside the termites is rather diverse, as there is a strong division of labour among them to accomplish the intricate process of lignocellulose digestion. The objectives of this work were to: 1) investigate the changes in flagellate protists communities of the termite Reticulitermes grassei in different locations; 2) test the possible effect of different laboratorial diets on diversity and abundance of the flagellate protists. R. grassei termites were captured in four different locations (Évora, Faial Island, Leiria and Sesimbra), in Portugal, and their symbiotic flagellate protist community diversity and abundance was evaluated. Termites belonging to the same colony were submitted to six different diets (natural diet, pine wood, European beech, thermally modified beech, cellulose and starvation) and after the trials their flagellate protist community was also evaluated. The differences between termite colonies from different locations may not be denied, although not considered to be significant. Similar flagellate protists communities were found on non-treated sound woods, while cellulose fed and starving termites had significantly different communities. The flagellate protists community of untreated beech and thermally modified beech fed termites were considered to be significantly different, with three morphotypes missing in the treated wood fed termites. Although the effects of geographical location were not considered significant, the laboratory diets caused major adaptations of the flagellate protists communities. The termite symbiotic flagellate protists community is a dynamic assemblage able to adapt to different conditions and diets.
S Duarte, M Duarte, P A V Borges, L Nunes


Response of the symbiotic flagellate protists community of subterranean termites to sublethal amounts of biocides
2018 - IRG/WP 18-10911
Subterranean termites are quite efficient at extracting nutrients from lignocellulose. Their ability relies not only on the digestive tract physiology but also on symbiotic relationships established with flagellate protists and bacteria. This work aimed to screen the response of the flagellate protists community of the subterranean termite Reticulitermes grassei Clément to the ingestion of different biocides. The substances chosen were applied at sublethal doses and included antibiotics (amoxicillin), an antiprotozoal (metronidazole), a termite intestine pH alteration agent and respiration inhibitor (boric acid), an essential oil (cloves) and its main constituent (eugenol), together with the solvent (water) and a positive control of pine wood. Termites were captured in three different zones of the same pine forest, sufficiently distant to be considered as different colonies; three replicate samples from each colony were selected for testing. Immediately after termite capture the initial flagellate protists community was evaluated for all samples (initial controls). Groups of termite workers were then fed on diet disks impregnated with the substances and, after the trials the diversity and abundance of the flagellate protist community was evaluated. Twelve morphotypes were present in the controls. The naturally less abundant morphotypes were positively associated with the termites screened before the trials and the ones fed on water treated diet disks or original wood. Metronidazole showed to affect negatively most morphotypes, however, two morphotypes’ abundance increased; these two morphotypes abundances decreased when termites fed on amoxicillin treated diet disks. For eugenol and boric acid significant negative impact was found for one morphotype with parallel increase in abundance of two others. Overall, the results suggest a possible maintenance of hindgut equilibrium or minimum functioning relying both on: changes on abundances of two or three morphotypes; and presence and abundance of the less common morphotypes. Three morphotypes exhibit differentiated response to changes in hindgut conditions, triggered by the addition of substances to the termite diet. This dynamic nutritional symbiosis equilibrium seems to provide a wide range of defences of the termite to exposure to substances potentially harmful and general dietary changes.
S Duarte, T Nobre, P Borges, L Nunes


Biological resistance of phenol-resin treated wood
1990 - IRG/WP 3602
Biological resistance of PF (phenol formaldehyde resin) - treated wood has been tested in relation to the resin properties, wood species and biological factors. When tested using water-soluble PF (mol. wt. 170), ca. 10% RI (resin impregnation) was enough to suppress the decay of Japanese cedar (Cryptomeria japonica) and western hemlock (Tsuga heterophylla) blocks exposed to Tyromyces palustris (brown-rot type) and Coriolus versicolor (white-rot type). For a decay suppression of Japanese beech (Fagus crenata) by treating with the same PF, ca. 20% RI was required for both cases of exposure. When using ethanol-soluble PF (mol. wt. 300), the lesser effect on decay suppression was revealed for most of wood-fungus combinations, suggesting a possible better penetration of lower molecular resin into the wood cell walls. PF treatment of wood also affected the termite Coptotermes formosanus, causing the severe depletion of feeding activity and the higher mortality at 5-15 (%) RI. Of the three species of symbiotic protozoa, the most cellulolytic Pseudotrichonympha grassii diminished first shortly after feeding.
M Takahashi, Y Imamura


The rôle of lignin in the nutrition of several Australian termites
1983 - IRG/WP 1191
The ability of Nasutitermes exitiosus (Hill), Coptotermes acinaciformis (Frogatt), Coptotermes lacteus (Froggatt) and Mastotermes darwiniensis (Froggatt) to degrade 14C-lignin preparations was examined. The lower termites were unable to degrade lignin. Nasutitermes exitiosus was able to cause a 5-8% degradation of hardwood lignins and a synthetic lignin. It failed to degrade the lignin of Pinus radiata D.Don.
L J Cookson


Distribution of the three symbiotic protozoa in Formosan subterranean termite, Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae)
1993 - IRG/WP 93-10010
Six colonies (three each from laboratory and field) of Formosan subterranean termite, Coptotermes formosanus Shiraki, were served for investigating the abundance and distribution of three symbiotic protozoa in the hindgut of workers. The total protozoan number amounted to 6,000-10,000 per a worker, and the order of the abundance of the three protozoa and the proportional distribution of each species in the hindgut were common among the colonies. Pseudotrichonympha grassii Koidzumi was the smallest in number (800-2,200 per a worker) and was preferentially distributed in the anterior part of the hindgut. Holomastigotoides hartmanni Koidzumi was medial in number (1,200-3,000), and the distribution was relatively uniform all through the hindgut. Spirotrichonympha leidyi Koidzumi was the most abundant in number (2,800-5,000) and was found mainly in the posterior part. These results appeared to support that the prominent localization of each protozoan species in the worker´s hindgut could be related to the nutritional metabolism in Coptotermes formosanus.
T Yoshimura, K Tsunoda, M Takahashi


Ingestion of wood-degrading micro-organisms
1991 - IRG/WP 4169
An ultrastructural study was carried out on the digestive tract contents of Limnoria lignorum, frass, gallery walls and surface structures of the animals. The purpose of the study was to determine whether wood-degrading bacteria, fungi and other microbes and/or wood degraded by these microbes were present in gut regions and therefore could provide indirectly a nutritional source for Limnoria. Both bacterial (tunnelling) and fungal (soft rot) degraded wood fragments as well as lysed microbes, extracellular slime and various other microbes (actinomycetes, diatoms) were found in fore, mid and hind gut regions. The range of microbes associated with wood fragments from gut regions were similar to those recognized either colonizing or degrading surrounding gallery walls or present on the external surfaces of the organism itself. Wood materials in the gut were highly fragmented and showed evidence for either extensive bacterial or soft rot attack, or surface and sub-surface evidence for carbohydrate removal. The major wood degrading bacteria attack noted was tunnelling bacteria as well as a form capable of cell wall dissolution of pine wood. Both bacterial types carried out extensive attack of pine wood fragments present in Limnoria tunnels. Lysed fungal hyphae, and soft rot hyphae associated with residual soft rotted wood fragments were also prominent. Gut regions of Limnoria lignorum lacked a natural resident bacteria flora, although numbers of characteristic Gram-negative active vesicle producing bacteria were represented in all gut regions associated with wood fragments. In hind gut regions a prominence of wood middle lamella regions was noted suggesting that these cell wall regions were more resistant to attack. Results indicate ingestion of bacterial and fungal degraded wood and its associated microflora. It is suggested that ingestion of microbial degraded wood could provide Limnoria with increased substrate assessibility and a greater surface area over which their enzymes could act. Associated bacterial and fungal breakdown products could also provide an important supplimentary source of nitrogen.
G F Daniel, S M Cragg, T Nilsson


The effect of boric acid on the protozoan numbers of the subterranean termite, Reticulitermes lucifugus
1996 - IRG/WP 96-10148
Lower termites harbor in their guts populations of microorganisms known to be indispensable for their survival, being responsible, at least partially, for the digestion of cellulose, the main item of termites'diet. The Reticulitermes species in particular harbor in their hindguts some unique intestinal fauna, for instance protozoa belonging to the genera Pyrsonympha, Dinenympha and Trychonympha. The effect of boric acid on these organisms was evaluated by direct enumeration of the total number of protozoa and the number of most relevant protozoa genera. Although there is a clear effect of the boron on the protozoan populations, the actual decrease in total numbers is not very great, and only one group of the larger protozoa (Trychonympha) tended to disappear with the increasing concentrations of active ingredient.
L Nunes, D J Dickinson


Detrimental effects of boric acid on symbiotic protozoa in Reticulitermes flavipes (Kollar) and Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae)
2000 - IRG/WP 00-10366
In laboratory choice bioassays, Reticulitermes flavipes (Kollar) and Coptotermes formosanus Shiraki were offered a tunneling soil consisting of boric acid (BA) mixed with sterilized soil at concentrations of 0.05, 0.25, 0.50, 1.00, 2.00, or 4.00 percent AI (wt:wt). Termites could choose to remain in their main nest that contained non-treated artificial substrate and adequate food, or tunnel through BA-treated soil in an attached foraging tube to reach a satellite nest that also contained non-treated artificial substrate and additional food. Gut protozoa populations were determined after 12 weeks. Termite tunneling through BA -treated soil resulted in moderate reduction to complete loss of symbiotic gut protozoa in both termite species as BA increased to the greatest concentration. Reductions in protozoa were most noticeable in the 2.00 and 4.00 percent BA concentrations. Boric acid was not repellent and termites removed BA-treated soil from foraging tubes and deposited it in main and satellite nests. Generally, at BA concentrations of 1.00-2.00% or less in soil, termite gut protozoa populations did not appear to be dose dependent. The four primary protozoa genera in R. flavipes and the three primary protozoa genera in C. formosanus were all detrimentally affected by exposure to BA. Overall, BA mixed in soil caused significant loss of protozoa that was very detrimental to both termite species.
B M Kard


The functional anatomy of the digestive caecae and gut residence times for the marine wood borer Limnoria (Crustacea: Isopoda)
1997 - IRG/WP 97-10204
A scanning and transmission electron microscope study of the digestive caecae of the marine wood boring isopods Limnoria tripunctata and Limnoria quadripunctata, was undertaken. Two cell types were identified, containing structures indicative of absorption, secretion and storage. Cell types were distinguished according to size, the largest cell type having a large free surface area, a well developed microvillous border and a cytoplasm containing both iron and what were believed to be proteinaceous granules. The small cells possessed endoplasmic reticulum and lipid granules. This study suggests that large cells were responsible for absorption and storage of iron, whereas the small cells were secretory and capable of storing lipid. Residence time for wood in the gut was determined to be approximately two hours for both Limnoria tripunctata and Limnoria quadripunctata. This rapid gut transit time is relatively short compared to other cellulose digesters, suggesting that Limnoria is an inefficient digester of wood.
C Wykes, S M Cragg, A J Pitman


Transfer of Termiticidal Dust Compounds and their Effects on Symbiotic Protozoa of Reticulitermes flavipes (Kollar)
2008 - IRG/WP 08-10661
Dusting of termites in situ has been used as a control measure for decades; however environmental awareness of the toxicity of certain compounds now limits their use (eg arsenical dusts). Our laboratory is in the process of suppressing an isolated colony of Reticulitermes flavipes (Kollar) from a small village of 250 homes in mid-central Wisconsin (Endeavor, WI). Initial treatment of the colony involved the use of 200 Microgen bait cartridges containing 0.25% diflubenzuron. The presence of many living termites occupying bait stations throughout the season prompted the addition of various dusting compounds, such as boron, N-N’naptahaloylhydroxylamine (NHA) and other dusts to aide in colony elimination. This laboratory study explores the efficacy of various insecticidal dusts to kill termites as well as to transfer the insecticide to nest mates. Mortality after primary (direct) dusting was found to occur with sodium borax, zinc borate, concrobium polymer (CP) and NHA in 7, 9, 9 and 21 days respectively. However, the ability to cause mortality to 25 un-dusted termites was impaired in the concrobium group and delayed in others. Further tests were performed to determine the mechanism of transfer by examining the survival rates of the symbiotic flagellated protists in the termite hindgut over time after dusting to determine if termiticidal dusts were killing the protozoa necessary for cellulose digestion. Possible mechanisms of mortality are discussed as well as the potential of using dusting compounds in termite treatment or eradication.
F Green III, R A Arango, G Esenther


Changes in bacterial gut community of Reticulitermes flavipes (Kollar) and Reticulitermes tibialis banks after feeding on termiticidal bait material
2014 - IRG/WP 14-10819
In this study, 454-pyrosequencing was used to evaluate the effect of two termiticidal baits, hexaflumuron and diflubenzuron, on the bacterial gut community in two Reticulitermes flavipes colonies and one Reticulitermes tibialis colony. Results showed two bacterial groups to be most abundant in the gut, the Bacteroidetes and Spirochaetes, both of which do not appear to be adversely affected by bait treatment according to analysis conducted to date. Other major bacterial lineages present included Actinobacteria, Fibrobacteres, Firmicutes, Fusobacteria, Proteobacteria, Tenericutes, TM7, Verrucomicrobia and unclassified species, which matches closely with other studies examining termite gut bacteria. Phylogenetic analysis examining similarity among treated groups versus controls showed a treatment effect in both R. flavipes colonies, but no effect on R. tibialis samples. Overall community analysis also showed treatment groups were separated by their collection location indicating a distinct bacterial community within a colony. Future analysis will focus on the types of bacteria affected by bait treatment and the role of these changes in overall termite fitness.
R A Arango, F Green III, K F Raffa


Diversity of hindgut symbiotic flagellate protist communities of the European subterranean termite in Portugal
2016 - IRG/WP 16-10875
The flagellate protist communities are an important part of the termite, as they lead the lignocellulose digestion. Termites (Reticulitermes grassei) were sampled from forest and urban environments in mainland Portugal where they are native and in Faial Island, Azores (invasive populations). Termites’ gut contents was analysed morphologically and the diversity of the flagellate protist community evaluated based on morphotypes. From the two Azorean invasive populations we were able to identify 12 different morphotypes whereas some of the populations in the mainland had as few as 6. Indeed, on the fourteen native populations the number of flagellate protists morphotypes ranged between 6 and 12. Shannon Wiener diversity index was used to calculate a variation partitioning between geographical and local variables. Our results suggest the existence of a core group of flagellate protists, probably performing key steps in the lignocellulose digestion. However, these communities may be more diversified and factors linked with the geographic location are likely a key influence of the flagellate protist communities analysed. In the invasive urban termite populations the high flagellate protist communities’ diversity, as well as the similarity between the two populations captured, may indicate a switch of R. grassei foraging and social habits in the invasive termite populations. Local conditions also influenced the flagellate protist communities, although not so markedly as geographic location. In this study, native termite colonies from urban environments showed the less diverse flagellate protist communities.
S Duarte, T Nobre, M Duarte, P A V Borges, L Nunes


Evaluating the role of Actinobacteria in the gut of wood-feeding termites (Reticulitermes spp.)
2017 - IRG/WP 17-10880
Nitrogen has been shown to be a limiting nutrient across a range of xylophagous insects. These insects often rely on symbiotic microorganisms in the gut for nitrogen acquisition, via fixation of atmospheric nitrogen or break down of other available nitrogenous substances. In phylogenetically lower, wood-feeding termites, the role of nitrogen fixing bacteria has been well studied. However, there is also evidence that uric acid can be metabolized into ammonia and serve as an additional nitrogen source. In this study, 36 Actinobacterial isolates (Streptomyces spp.) from the guts of Reticulitermes flavipes (Kollar) and Reticulitermes tibialis Banks, were screened for uric acid breakdown using culture-based methods. Results showed 92% of isolates are capable of degrading uric acid, with 35% classified as having “very strong” uricase activity in vitro. Enzyme assays of four representative Actinobacterial isolates confirmed that uric acid was broken down and ammonia was produced. Soil materials manipulated by termites also showed increased uricase activity compared to soil alone. However, this increase was not accompanied by an increase in overall abundance of Actinobacteria. It is still possible, however, that only those Actinobacteria with uricase activity increase while others remain the same or decrease, which would not change overall abundance values. Results from this study support the hypothesis that Actinobacteria associated with the gut of wood-feeding termites have the potential to contribute to nitrogen acquisition via uricolysis. Future work will be aimed at better understanding this complex relationship between wood-feeding subterranean termites and gut-associated Actinobacteria.
Evaluating the role of Actinobacteria in the gut of wood-feeding termites (Reticulitermes spp.)


Application of nano metal fluorides against termites
2019 - IRG/WP 19-30733
Recent research has shown that fluorides such as NaF have potential as a wood preservative against termite attack (Pan and Wang, 2015). However, NaF has high leaching susceptibility because of its solubility (40 g/l ) which can reduce its efficacy over long term. Metal fluoride nanoparticles such as MgF2 and CaF2 present a viable alternative to water soluble fluoride-based wood preservatives as they have low water solubility. The solubility of MgF2 is 130 mg/l and that of CaF2 is 16 mg/l. It is found that synthesis of low-water soluble fluorides as nanoparticles allow them to be introduced into wood specimens. These homo-dispersed nano metal fluorides are synthesized as sols using fluorolytic sol-gel synthesis (Krahl et al. 2016). Wood specimens treated with fluoride sols of MgF2 and CaF2 have been tested against subterranean termites (Coptotermes formosanus) in accordance with EN 117. Coptotermes formosanus is a species that causes significant decay of wood especially in Taiwan, Japan, and southern United States (Chang and Cheng 2002). After eight-week testing according to EN 117, the specimens treated with nano metal fluorides had an average rating of 1.2 while the control specimens had an average rating of 4. Moreover, after the eight-week test almost all termites exposed to treated specimens were dead, while the termites exposed to untreated specimens had average 55% survival. Thus, the nano metal fluoride treated wood specimens were toxic to termites leading to the observed high mortality and low rating. A preliminary analysis of the protists in the gut of the termites exposed to nano metal fluoride treated wood specimens found that the number of gut protists were lower than those exposed to untreated specimens.
S M Usmani, K Klutzny, Y de Laval, R Plarre, D McMahon, I Stephan, T Hübert, E Kemnitz