IDH knockdown alters foraging behavior in the termite Odontotermes formosanus in different social contexts

Foraging, as an energy-consuming behavior, is very important for colony survival in termites. How energy metabolism related to glucose decomposition and adenosine triphosphate (ATP) production influences foraging behavior in termites is still unclear. Here, we analyzed the change in energy metabolism in the whole organism and brain after silencing the key metabolic gene isocitrate dehydrogenase (IDH) and then investigated its impact on foraging behavior in the subterranean termite Odontotermes formosanus in different social contexts. The IDH gene exhibited higher expression in the abdomen and head of O. formosanus. The knockdown of IDH resulted in metabolic disorders in the whole organism. The dsIDH-injected workers showed significantly reduced walking activity but increased foraging success. Interestingly, IDH knockdown altered brain energy metabolism, resulting in a decline in ATP levels and an increase in IDH activity. Additionally, the social context affected brain energy metabolism and, thus, altered foraging behavior in O. formosanus. We found that the presence of predator ants increased the negative influence on the foraging behavior of dsIDH-injected workers, including a decrease in foraging success. However, an increase in the number of nestmate soldiers could provide social buffering to relieve the adverse effect of predator ants on worker foraging behavior. Our orthogonal experiments further verified that the role of the IDH gene as an inherent factor was dominant in manipulating termite foraging behavior compared with external social contexts, suggesting that energy metabolism, especially brain energy metabolism, plays a crucial role in regulating termite foraging behavior.     

Occurrence and metabolic role of the pyruvate dehydrogenase complex in the lower termite Coptotermes formosanus (Shiraki)

The activities of the pyruvate dehydrogenase complex in extracts of the gutted body, head, foregut/midgut and hindgut (hindgut epithelium and microorganisms) tissues of the lower termite Coptotermes formosanus (Shiraki) were determined by measuring the [¹⁴C]-acetyl-CoA produced from [2-¹⁴C]-pyruvate and the ¹⁴CO₂ produced from [1-¹⁴C]-pyruvate. The activities of pyruvate dehydrogenase, l-lactate dehydrogenase, acetyl-CoA synthetase, malate dehydrogenase (decarboxylating), and acetate kinase in the termite tissues and the hindgut also were determined. The sum (7.1 nmol/termite/h) of the pyruvate dehydrogenase complex activities in the termite tissues other than the hindgut was five times higher than the activity in the hindgut. Significant amounts of l-lactate dehydrogenase activity were found in all of the tissues. All of the tissues other than the hindgut had significant amounts of acetyl-CoA synthetase activity. Malate dehydrogenase (decarboxylating) activity was about ten times higher in the hindgut extract than in the gutted body and head extracts and the activity in the foregut/midgut extract was very low. These results indicate that acetyl-CoA for the TCA cycle is produced effectively in the tissues of the termite itself, both from pyruvate by the pyruvate dehydrogenase complex and from acetate by acetyl-CoA synthetase.     

A novel approach to characterize branching network: Application to termite tunnel patterns

We defined a novel “branch length similarity” (BLS) entropy, S, on a simple network consisting of a single node and branches. This simple network is referred to as “unit branching network” (UBN) because UBNs are components of larger networks. As an application of BLS entropy, we considered the characterization of termite tunnel patterns because termite tunnel patterns can be broken down into a collection of simple units consisting of a single node and branches. These simple units correspond to UBNs. To this end, in additional to the entropy, we introduced the standard deviation (σ) of the difference in S between UBNs connected by a single tunnel branch. Forty simulated tunnel patterns were created for each of two termite species, Reticulitermes flavipes (Kollar) and Coptotermes formosanus Shiraki. These patterns were projected into <S>–σ phase space in order to assess their topological properties. This approach showed that for R. flavipes, their coordinates were relatively more clustered than those of C. formosanus. This result reflected that these two species were differently constrained by emergent property resulting from simple worker's tunneling behavior. We believe that the approach proposed in this study can be a useful tool to explore termite tunnel systems, but not limited to termite system.     

The complete mitogenome of the Formosan termite, Coptotermes formosanus Shiraki

The Formosan termite Coptotermes formosanus Shiraki is a well-known invasive pest that causes severe damage to wooden structures in many parts of the world. Although several studies examined its phylogeographic patterns using a few mitochondrial genes, the phylogenetic relationships among C. formosanus are poorly understood because of the small number of mutations known among its mitochondrial genes. To provide a useful genetic tool for further analyses, we analyzed the complete mitochondrial genome sequence of C. formosanus using specimens collected from three isolated islands in the Ryukyu Archipelago of Japan. The circular mitogenome of these termites consisted of genes encoding 22 transfer RNAs, two ribosomal RNAs, and 13 mitochondrial proteins, as is the case for most animal mitochondrial genomes. The G + C content was 34.1%, and the total length varied slightly between 16,234 and 16,236 base pairs. The complete mitochondrial genomes of the three populations were more than 99.9% identical to each other and showed differences at six nucleotide positions. The COII, 12S rRNA, and 16S rRNA genes that are commonly used for phylogenetic analyses revealed only one substitution or no substitutions. The mitogenome sequences determined here should contribute to the design of new molecular markers for the clarification of the historical distribution process of C. formosanus and for further phylogenetic analyses with this and related termite species.     

Effects of phosphodiesterase inhibitors on glucose utilization in isolated cardiac myocytes

The phosphodiesterase (PDE) inhibitor, enoximone, enhances the oxidation of fatty acids in cardiac myocytes. Since carbohydrate oxidation is tightly coupled and inversely related in cardiac tissue to fatty acid oxidation, this study was designed to investigate enoximone's effects on glucose metabolism in the heart. To determine if enoximone alters this reciprocal relationship, the effects of enoximone on [U-¹⁴C]glucose and [2-¹⁴C]pyruvate oxidation were determined in isolated cardiac myocytes. The effect of PDE inhibitors was also examined on pyruvate dehydrogenase complex (PDH) activity, a key component of oxidative glucose metabolism. Two PDE inhibitors, enoximone and milrinone, decreased PDH activity by 69 and 64%, respectively at 0.5 mM. This inhibition of PDH activity by enoximone was completely reversed after removing enoximone from the myocyte medium. PDH activity was unaffected by agents which alter cyclic nucleotide signaling: cGMP, dibutyryl cyclic AMP, and AMP. The effect of enoximone on [2-¹⁴C]pyruvate oxidation was similar to that on PDH. Interestingly, the oxidation of glucose was decreased 35% by 0.5 mM enoximone. In isolated rat heart mitochondria (RHM), enoximone decreased PDH activity by 37%. These studies suggest that PDE inhibitors decrease carbohydrate utilization by inhibiting the PDH complex in the heart. The inhibition of PDH by PDE inhibitors appears unrelated to their effects on cAMP or cGMP. This inhibition of PDH by PDE inhibitors may occur, at least in part, secondary to stimulating fatty acid oxidation.     

Characterization of a laccase from a wood‐feeding termite,Coptotermes formosanus

Coptotermes formosanus Shiraki is a wood‐feeding termite which secretes a series of lignolytic and cellulolytic enzymes for woody biomass degradation. However, the lignin modification mechanism in the termite is largely elusive, and the characteristics of most lignolytic enzymes in termites remain unknown. In this study, a laccase gene lac1 from C. formosanus was heterogeneously expressed in insect Sf9 cells. The purified Lac1 showed strong activities toward hydroquinone (305 mU/mg) and 2,6‐dimethoxyphenol (2.9 mU/mg) with low K_m values, but not veratryl alcohol or 2,2’‐azino‐bis (3‐ethylbenzothiazoline‐6‐sulfonic acid). Lac1 could function well from pH 4.5 to 7.5, and its activity was significantly inhibited by H₂O₂ at above 4.85 mmol/L (P < 0.01). In addition, the lac1 gene was found to be mainly expressed in the salivary glands and foregut of C. formosanus, and seldom in the midgut or hindgut. These findings suggested that Lac1 is a phenol‐oxidizing laccase like RflacA and RflacB from termite Reticulitermes flavipes, except that Lac1 was found to be more efficient in phenol oxidation, and it did not require H₂O₂ for its function. It is suspected that this kind of termite laccase might only be able to directly oxidize low redox‐potential substrates, and the high redox‐potential groups in lignin might be oxidized by other enzymes in the termite or by using the Fenton reaction.     

Extended disease resistance emerging from the faecal nest of a subterranean termite

Social insects nesting in soil environments are in constant contact with entomopathogens but have evolved a range of defence mechanisms, resulting in both individual and social immunity that reduce the chance for epizootics in the colony, as in the case of subterranean termites. Coptotermes formosanus uses its faeces as building material for its nest structure that result into a ‘carton material’, and here, we report that the faecal nest supports the growth of Actinobacteria which provide another level of protection to the social group against entomopathogens. A Streptomyces species with in vivo antimicrobial activity against fungal entomopathogens was isolated from the nest material of multiple termite colonies. Termite groups were exposed to Metarhizium anisopliae, a fungal entomopathogen, during their foraging activity and the presence of Streptomyces within the nest structure provided a significant survival benefit to the termites. Therefore, this report describes a non-nutritional exosymbiosis in a termite, in the form of a defensive mutualism which has emerged from the use of faecal material in the nesting structure of Coptotermes. The association with an Actinobacteria community in the termite faecal material provides an extended disease resistance to the termite group as another level of defence, in addition to their individual and social immunity.     

Fungal nutrition allocation enhances mutualism with fungus-growing termite

Fungal nodules and aged fungus gardens are products of termite fungiculture systems, and are the diets of termites. To understand the nutrition flow in fungiculture, we quantified the number and mass of fungal nodules produced along with fungus garden maturation and analysed the α-amino acid and fatty acid compositions of fungal nodules, fungus gardens, and termite tissues of a fungus-growing termite, Odontotermes formosanus. 1 g of fungus garden produced 5,148 fungal nodules (∼68.0 mg). Approximately 7.0% of α-amino acids were allocated to the fungal nodules and the rest (∼93.0%) remained in the fungus gardens. The compositions of α-amino acids or fatty acids in aged fungus gardens and fungal nodules were more similar to that of termite tissues than fresh fungus gardens, which supports the idea that termites nutritionally depend on the fungal products. Among the 18 α-amino acids, tryptophan was an essential amino acid and was the only one missing from fresh and aged fungus gardens, but found in fungal nodules at significantly higher concentrations. Hence, termites must consume fungal nodules to obtain tryptophan for survival. Furthermore, the fungus spores incorporated in nodules, were transferred when nodules were ingested by termites. We propose that allocating tryptophan in fungal nodules is crucial to enhance the mutualism between the fungus and termite.     

Musty odor of entomopathogens enhances disease-prevention behaviors in the termite Coptotermes formosanus

Termites often eliminate pathogens directly through mutual grooming, and are thereby prevent infections from entomopathogenic fungi. Our previous study confirmed that the antennae of Coptotermesformosanus sensitively responded to the musty odor of entomopathogenic fungi. However, it is unclear if this odor has any effect on termite behavior. The purpose of this study was to clarify the effects of fungal odor on termite behavior, especially on conidia removal. The musty odor was prepared as an aqueous solution by immersing conidia in distilled water. When untreated termites were mixed with fungal-odor-treated termites at a ratio of 4:1, mutual grooming and attack of treated termites were frequently observed. This indicated that the fungal odor triggered these behavioral responses. While some components of the fungal odor were found in all of the entomopathogenic fungi tested, the odor profiles differed among the isolates.     

Disruption and mutagenesis of the Saccharomyces cerevisiae PDX1 gene encoding the protein X component of the pyruvate dehydrogenase complex

Disruption of the PDX1 gene encoding the protein X component of the mitochondrial pyruvate dehydrogenase (PDH) complex in Saccharomyces cerevisiae did not affect viability of the cells. However, extracts of mitochondria from the mutant, in contrast to extracts of wild-type mitochondria, did not catalyze a CoA- and NAD(+)-linked oxidation of pyruvate. The PDH complex isolated from the mutant cells contained pyruvate dehydrogenase (E1 alpha + E1 beta) and dihydrolipoamide acetyltransferase (E2) but lacked protein X and dihydrolipoamide dehydrogenase (E3). Mutant cells transformed with the gene for protein X on a unit-copy plasmid produced a PDH complex that contained protein X and E3, as well as E1 alpha, E1 beta, and E2, and exhibited overall activity similar to that of the wild-type PDH complex. These observations indicate that protein X is not involved in assembly of the E2 core nor is it an integral part of the E2 core. Rather, protein X apparently plays a structural role in the PDH complex; i.e., it binds and positions E3 to the E2 core, and this specific binding is essential for a functional PDH complex. Additional evidence for this conclusion was obtained with deletion mutations. Deletion of most of the lipoyl domain (residues 6-80) of protein X had little effect on the overall activity of the PDH complex. This observation indicates that the lipoyl domain, and its covalently bound lipoyl moiety, is not essential for protein X function. However, deletion of the putative subunit binding domain (residues approximately 144-180) of protein X resulted in loss of high-affinity binding of E3 and concomitant loss of overall activity of the PDH complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Components of cellulase from the higher termite,Nasutitermes walkeri

The cellulase from the termite Nasutitermes walkeri consists of two enzymes. Each has broad specificity with predominantly one activity. One enzyme is an endo-gb-1,4-glucanase (EC 3.2.1.4) which predominantly cleaves cellulose randomly to glucose, cellobiose and cellotriose. It hydrolyses cellotetraose to cellobiose but will not hydrolyse cellobiose or cellotriose. The second enzyme component is a β-1,4-glucosidase (EC 3.2.1.21) as its major activity is to hydrolyse cellobiose, cellotriose and cellotetraose to glucose; it has some exoglucosidase activity as glucose is the only product produced from cellulose. Its cellobiase activity is inhibited by glucono-δ-lactone.     

Distribution patterns of four Termitomyces species cultivated by a fungus-growing termite,Odontotermes formosanus,in Taiwan

Fungus-growing termites inoculate the obligate symbiotic fungus Termitomyces into the fungus comb in their colonies. In this study, Taiwanese Termitomyces species were determined by diagnostic PCR using the metagenome of the body of Odontotermes formosanus (the only host termite living in Taiwan) as a template. Phylogenetic analyses revealed that four different Termitomyces species are cultivated by O. formosanus in Taiwan. Three have previously been registered in the DNA database, but one was first recorded in Taiwan. Only Termitomyces sp. Type C was distributed in all areas investigated in Taiwan, whereas the other three species were distributed regionally. Field observations indicated that the flush period and the number of fruit bodies in each colony varied between species. The distribution patterns of Termitomyces spp. in Taiwan may be related to the Taiwanese climate and/or the fruiting pattern.     

The dihydrolipoamide dehydrogenase of Aeromonas caviae ST exhibits NADH-dependent tellurite reductase activity

Potassium tellurite (K₂TeO₃) is extremely toxic for most forms of life and only a limited number of organisms are naturally resistant to the toxic effects of this compound. Crude extracts prepared from the environmental isolate Aeromonas caviae ST catalize the in vitro reduction of in a NADH-dependent reaction. Upon fractionation by ionic exchange column chromatography three major polypeptides identified as the E1, E2, and E3 components of the pyruvate dehydrogenase (PDH) complex were identified in fractions exhibiting tellurite-reducing activity. Tellurite reductase and pyruvate dehydrogenase activities co-eluted from a Sephadex gel filtration column. To determine which component(s) of the PDH complex has tellurite reductase activity, the A. caviae ST structural genes encoding for E1 (aceE), E2 (aceF), and E3 (lpdA) were independently cloned and expressed in Escherichia coli and their gene products purified. Results indicated that tellurite reductase activity lies almost exclusively in the E3 component, dihydrolipoamide dehydrogenase. The E3 component of the PDH complex from E. coli, Zymomonas mobilis, Streptococcus pneumoniae, and Geobacillus stearothermophilus also showed NADH-dependent tellurite reductase in vitro suggesting that this enzymatic activity is widely distributed among microorganisms.     

Light alters activity but does not disturb tandem coordination of termite mating pairs

Group-living animals coordinate their movements via local interactions, which can be mediated by visual, tactile, and chemical communication channels. Termite mating pairs form tandems with one male imago following one female imago in a synchronised way to explore the environment and search for a nesting site. Imagoes are the only developmental stage with compound eyes in termites, but the role of vision during tandem runs remains unknown. Here, we investigate the movements during tandem runs of two termite species, Coptotermes formosanus, which swarms during the night, and Reticulitermes speratus, which swarms during the day. We performed the experiments with light and in complete darkness. We found that females and males of both species adjust their speed to each other to form a stable tandem and reunite efficiently upon separation, with or without light. However, the activity was dependent on light conditions in the diurnal R. speratus, in which termites were more active with light. On the other hand, the nocturnal C. formosanus was mostly insensitive to light environments, with termites being slightly more active in darkness. Our results suggest that termites can use light as an environmental cue to start forming mating pairs but not as means to locate mates or coordinate their movements.     

Formosan subterranean termite swarming behavior and alate sex-ratio (Isoptera: Rhinotermitidae)

Summary The Formosan subterranean termite (Coptotermes formosanus Shiraki) was first sighted in the cities of New Orleans and Lake Charles, Louisiana, in 1966. Its distribution in the historic French Quarter of New Orleans 23 years later (1989) and in 1991 and 1992 is reported here using counts of alates from light-traps. Light-trappings revealed a Quarter-wide distribution of the termite.C. formosanus shows an extended pause in flight behavior in the middle of the two-month flight season that is predictable and independent of weather. A colony-level mechanism, namely, developmental inhibition of nymphs by alates, may regulate this intermission in flight. The sex-ratio of light-trapped alates varies over time, but is generally female-biased, especially at the height of the flight season.     

Distribution pattern of Termitomyces types symbiotic with the fungus‐growing termite Odontotermes formosanus on Okinawa Island

Fungus‐growing termites (subfamily Macrotermitinae) cultivate the symbiotic basidiomycete fungus Termitomyces in their fungus comb to digest cellulosic materials and to supply nitrogen‐rich fungal diet. In Japan, the fungus‐growing termite Odontotermes formosanus is found on the Yaeyama Islands and Okinawa Island, Okinawa Prefecture. Odontotermes formosanus is thought to have been recently and artificially introduced to Okinawa Island as its distribution is discontinuous and restricted to small areas. Previous DNA analyses revealed that two types of Termitomyces, namely Termitomyces sp. Type A and Termitomyces sp. Type B, whose fruiting bodies correspond to Termitomyces microcarpus‐like pseudorhiza‐lacking small mushroom and Termitomyces intermedius, respectively, are cultivated by O. formosanus on the Yaeyama Islands. However, information about the Termitomyces types cultivated by O. formosanus on Okinawa Island is limited. To define the fungal types cultivated by O. formosanus on Okinawa Island, I developed a diagnostic polymerase chain reaction method using primer sets specific to the nuclear ribosomal DNA sequences consisting of the internal transcribed spacers (ITS1 and ITS2) and 5.8S rDNA of Termitomyces not using fungal mycelium, but using the termite gut metagenome including fungal DNA as a template. The results indicated that the same two Termitomyces types from Iriomote Island are cultivated by O. formosanus on Okinawa Island. The distribution pattern of Termitomyces types on Okinawa Island showed that Termitomyces sp. Type A is limited to the mountainous side of Sueyoshi Park, despite Termitomyces sp. Type B being widely distributed in the area in which O. formosanus is found. This finding implies that O. formosanus on Okinawa Island was recently introduced from Iriomote Island to Sueyoshi Park.     

Testing the role of cuticular hydrocarbons on intercolonial agonism in two subterranean termite species (Coptotermes) and their hybrids

Recognition of nestmates is an important function in many social insects, as it maintains colony integrity by preventing outsiders from entering the colony. Agonism usually results from the interaction of con-specific non-nestmate individuals in termite colonies. Previous studies hypothesized that the cuticular hydrocarbon (CHC) profile of individuals had a role in nestmate recognition. However, contradictory results from previous studies in some subterranean termites raise questions on the validity of the cuticular hydrocarbon hypothesis. In the current study, Coptotermes gestroi (Wasmann), Coptotermes formosanus Shiraki and their hybrids were reared in identical conditions from colony foundation. This approach eliminates sources of variability in their cuticular hydrocarbon profiles aside from a genetic component. The parental species displayed dissimilar profiles of predominant alkanes and methyl alkanes, but both hybrid types displayed an overlapping, intermediate profile of these CHC. The mixture of the most abundant CHCs alone did not determine kin recognition; while the two hybrid types’ CHC profiles converged, the hybrids still showed strong agonism. One of the hybrid mating types easily merged with C. formosanus, despite only partial genetic similarity and dissimilar cuticular profiles for the common alkanes and methyl alkanes. This study suggests that in Coptotermes termites, the variable abundance of the major alkanes and methyl alkanes commonly found in most Coptotermes species does not explain agonistic patterns, and that other factors such as possibly more complex but less abundant CHC are likely to be involved in colonial recognition.

     

Comparative study of incipient colony development in the Formosan subterranean termite, <Emphasis Type="Italic">Coptotermes formosanus</Emphasis> Shiraki (Isoptera,Rhinotermitidae)

Summary The Formosan subterranean termite, Coptotermes formosanus Shiraki is the most destructive, difficult to control and economically important species of termite in the southern United States. At present, no information is available on the genetic relatedness of primary Formosan subterranean termite reproductives that establish new colonies. Information on survivorship and fitness components of primary reproductives from different sibships (sibling or nonsibling) is helpful to our understanding of biological and ecological characteristics of different breeding generations in C. formosanus. The present study examined the effects of sibship and colony origin on growth and mortality of incipient colonies of C. formosanus. Seven stock colonies of C. formosanus were collected in 1996 through 1997 in New Orleans and Lake Charles, La, USA. A total of 338 incipient colonies of sibling pairs or nonsibling pairs of C. formosanus were set up. The study indicated that mate relatedness significantly affected mortality and fitness. Nonsibling mates suffered significantly higher mortality than sibling mates originated from New Orleans. However, the decreased success of outbred mates was offset by an increased fecundity compared to inbred colonies over time. Both sibling- and nonsibling-founded colonies from Lake Charles had a significantly higher survival rate than did colonies from New Orleans. Colonies from Lake Charles also produced a significantly higher number of larvae/workers than colonies from New Orleans. The mismatch of habits by mates from different locations and the potential for greater disease risks may be associated with higher mortality in outbred pairs. However, heterozygous offspring of outbred pairs probably have increased genetic variation, which provides greater adaptation potential, thus making the colony more robust in the face of environmental fluctuations.Received 11 March 2002; revised 26 February 2003; accepted 14 March 2003.     

The P2X7 receptor is a key modulator of aerobic glycolysis

Ability to adapt to conditions of limited nutrient supply requires a reorganization of the metabolic pathways to balance energy generation and production of biosynthetic intermediates. Several fast-growing cells overexpress the P2X7 receptor (P2X7R) for extracellular ATP. A feature of this receptor is to allow growth in the absence of serum. We show here that transfection of P2X7R allows proliferation of P2X7R-transfected HEK293 (HEK293-P2X7) cells not only in the absence of serum but also in low (4 mM) glucose, and increases lactate output compared with mock-transfected HEK293 (HEK293-mock) cells. In HEK293-P2X7, lactate output is further stimulated upon addition of exogenous ATP or the mitochondrial uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP). In the human neuroblastoma cell line ACN, lactate output is also dependent on P2X7R function. P2X7R-expressing cells upregulate (a) the glucose transporter Glut1, (b) the glycolytic enzymes glyceraldehyde 3-phosphate dehydrogenase (G3PDH), (c) phosphofructokinase (PFK), (d) pyruvate kinase M2 (PKM2) and (e) pyruvate dehydrogenase kinase 1 (PDHK1); furthermore, P2X7R expression (a) inhibits pyruvate dehydrogenase (PDH) activity, (b) increases phosphorylated Akt/PKB and hypoxia-inducible factor 1α (HIF-1α) expression and (c) enhances intracellular glycogen stores. In HEK293-P2X7 cells, glucose deprivation increases lactate production, expression of glycolytic enzymes and ph-Akt/PKB level. These data show that the P2X7R has an intrinsic ability to reprogram cell metabolism to meet the needs imposed by adverse environmental conditions.