Termites possess both humoral and cellular immunity reviewed in . Pioneering work by, e. A number of genes and peptides related to this kind of humoral immunity have been described in termites. Among them are a transferrin gene which was up-regulated following exposure to an entomopathogenic fungus  , termicin, spinigerin  and gram-negative binding proteins GNBPs which can act as both PRRs and AMPs in several termite species .
Production of AMPs appears to differ not only between species  , but also between termites belonging to different colonies or castes  ,  , . The components of cellular immunity in termites are less well studied although melanization and encapsulation of foreign bodies have been frequently used as bioassay for immune competence e. While both, detoxification enzymes as well as immune proteins have been detected in termites, full genome screening and gene expression studies are still in their infancy where termites are concerned.
We chose the Formosan subterranean termite FST , Coptotermes formosanus Shiraki Blattodea: Rhinotermitidae , as model to investigate constitutive and induced gene expression related to xenobiotic metabolism and immunity in termites. This termite represents one of the most successful and economically important invasive pest species worldwide  , .
As a subterranean termite, C. Colonies are densely populated and can contain over a million individuals . Such a life style should be conducive for growth and spread of pathogens, however, 50 years of failed biocontrol efforts  suggests the existence of strong synergistic immune and detoxification defense mechanisms of subterranean termite colonies. Annotation of our previously constructed expressed sequence tag library of the FST revealed the presence of putative detoxification and immune-related genes . Out of those, we selected the 10 genes with the most similarity to known detoxification genes e.
A recent study aiming at a more complete description of the immune related transcriptome in C. However, colony or caste specific gene expression, inducible detoxification or possible crosslinks between genes involved in immunity and xenobiotic metabolism were not considered.
In fact, very few studies, have considered differential gene expression of immunity and detoxification genes among castes of termites  , . All but two target genes were inducible, either with a sublethal dose 0. Only one of the cytochrome P genes CYP and the gene for Down syndrome cell adhesion molecule Dscam were not induced by either treatment. Most genes were inducible by both treatments. The gene encoding for glutathione S-transferase GST was exclusively induced by 0. The y-axes denote relative expression in R units averaged over three colonies note that the ranges of y-values vary across graphs.
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Tests for variance between colonies and for interactions between colony and treatment or between colony and caste showed no significant effect of colony origin at any of the 10 target genes. Probabilities of colony variability and interaction ranked from 0. Thus, regardless of treatment or caste, R values were not significantly different among the three termite colonies.
In contrast to colony origin, treatment type 0. In contrast, GST was exclusively induced after 0.
Furthermore, MatE was only induced in workers by septic injury. GST was also exclusively induced in workers, but only after challenge with 0. Constitutive expression of all the investigated genes in FST workers and soldiers was low; however, eight out of the ten genes were inducible by one or both treatment types in one or both castes. Treatment consisted of exposure to a xenobiotic substance 0.
Relative levels of caste workers vs. Genes are arranged from left to right according their putative function ranging from detoxification to immunity and from top to bottom according to their caste dependent expression. The barbiturate PB is used to treat epilepsy in humans and acts on GABA-gated chloride channels in the central nervous system to reduce excitatory synaptic response similar in activity to some termiticides, i.
Response elements and nuclear receptors responsible for induction of many cytochrome Ps and GSTs have been described in insects and shown to respond to several groups of xenobiotics, including PB . In insects the GSTs are known to metabolize plant allelochemicals and insecticides, and high levels of GSTs are related to insecticide resistance.
Exposure to PB has been previously shown to induce GST activity in cockroaches  , Drosophila  ,  ,  , Aedes aegypti  , among others reviewed in . The lack of upregulation in soldiers and the effect of caste on differential gene expression are discussed below. Although, cytochrome P enzymes are largely ubiquitous and have common catalytic mechanisms, they are translated from a large family of genes with high sequence diversity, which explains their numerous functions.
In insects, cytochrome Ps are known to play a role in detoxification of insecticides and xenobiotics reviewed in  , but also in regulation of caste development via metabolism of hormones, like the juvenile hormone JH  ,  , . The product of CYP15A1 epoxidizes methyl farnesoate to juvenile hormone III in cockroaches  ,  and a similar function in JH biosynthesis was suggested in termites  , . Tarver et al. However, this study  did not investigate whether these two genes were functionally related.
Unlike other prominent cytochrome Ps, which metabolize a variety of pesticides due to their broad substrate and product specificity, CYP15A1 was shown to be highly specific in the silkworm .
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The CYP15A1 gene in our study was the only target gene that was constitutively expressed and induced by both treatment types in workers and soldiers. The fundamental role in JH biosynthesis and caste regulation of similar genes in termites  may explain the level of constitutive expression observed in our study.
The inducibility by PB and septic injury, however, suggests that the CYP15A1 gene product has expanded functionality in terms of xenobiotic metabolism and immunity beyond its primary function as epoxidase catalyzing the last step in JH biosynthesis. While PB inducibility of CYPs of the families 4, 6, 9, 12 and 28 in insects is well known, the only inducers for CYP15 family members so far reported were JH and terpene components from soldier head extracts suspected to act as caste regulatory primer pheromones .
Our study is the first to demonstrate inducibility of a member of the CYP15 family by a xenobiotic. However, the involvement of the CYP15 enzyme in actual xenobiotic metabolism and detoxification has to be confirmed in enzyme and bioassays. A link between JH levels and innate immunity has been discovered in the silkworm Bombus mori  , when JH treatment acted as an immune activator and increased the transcription products of six AMPs. We suggest that septic injury in our study upregulated CYP15A1 expression in termite workers and soldiers, which results in JH biosynthesis to provide increased JH levels as an immune activator.
This route would constitute an immune pathway different but complementary to the Toll- and Imd pathways.
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However, the investigated CYPs and GST genes were not constitutively expressed at elevated levels in Formosan termites and induction with PB led to a rather moderate spike in gene expression less than 4 fold , which is in the range of susceptible insects . Insecticide resistant strains usually show a much higher fold-change . Although there is variability in tolerance to insecticides among termite colonies and species, to date, no resistance or treatment failure has been recorded.
The second cytochrome P gene in the study CYP did not respond to treatment at all. A similar gene CYP12A1 in the house fly was shown to be inducible by PB and to metabolize different insecticides . Thus, we initially hypothesized that this gene would be most likely up-regulated in response to PB treatment and possibly involved in xenobiotic metabolism. Genome-wide studies in other insects showed that only a third of CYP genes are inducible by xenobiotics .
They function as transmembrane pumps and are involved in the excretion of xenobiotics in vertebrates, detoxification of metals and secondary metabolites in plants  and mediating resistance to antibiotics in bacteria  , . It was somewhat unexpected that MatE expression was upregulated by septic injury and not by xenobiotic challenge in our study.
The only evidence to date that MatE might be involved in antimicrobial defense is found in plant studies. Nawrath et al.
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To our knowledge, MatE proteins and their function in insects have not yet been investigated. It is possible that the MatE gene in our study was directly involved in immune response in termites as indicated by the induced expression in FST workers injected with E. However, it is more likely that MatE was upregulated as a consequence of the production of cytotoxic metabolites resulting from the activation of immune pathways by bacteria injection. For example, septic injury in insects activates the PPO pathway, which produces melanin, a phenolic biopolymer.
Some melanin precursors are cytotoxic, which is advantageous for killing opportunistic invaders at the wound site .
However, metabolites might be inadvertently cytotoxic to the insect itself, especially since the PPO cascade is taking place in the open circulatory system and thus byproducts may accumulate in the hemolymph. MatE proteins play an important role in the removal of metabolic waste products and are believed to be a transport system for phenolic compounds . Excessive melanin or its intermediaries from the PPO pathway might thus be extruded or sequestered by MatE in termites.
We initially assumed that the chitin binding protein CBP belongs into the category of immune related genes.
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