M. Slaytor

Evidence from multiple gene sequences indicates that termites evolved from wood-feeding cockroaches

Despite more than half a century of research, the evolutionary origin of termites remains unresolved [1] [2] [3]. A clear picture of termite ancestry is crucial for understanding how these insects evolved eusociality, particularly because they lack the haplodiploid genetic system associated with eusocial evolution in bees, ants, wasps and thrips [4] [5]. Termites, together with cockroaches and praying mantids, constitute the order Dictyoptera, which has been the focus of numerous conflicting phylogenetic studies in recent decades [6] [7] [8] [9] [10] [11] [12]. With the aim of settling the debate over the sister-group of termites, we have determined the sequences of genes encoding 18S ribosomal RNA, mitochondrial cytochrome oxidase subunit II (COII) and endogenous endo-beta-1, 4-glucanase (EG) from a diverse range of dictyopterans. Maximum parsimony and likelihood analyses of these sequences revealed strong support for a clade consisting of termites and subsocial, wood-feeding cockroaches of the genus Cryptocercus. This clade is nested within a larger cockroach clade, implicating wood-feeding cockroaches as an evolutionary intermediate between primitive non-social taxa and eusocial termites.

Cellulose digestion in termites and cockroaches: What role do symbionts play?

Abstract 1. 1. Termites and cockroaches are excellent models for studying the role of symbionts in cellulose digestion in insects: they eat cellulose in a variety of forms and may or may not have symbionts. 2. 2. The wood-eating cockroach, Panesthia cribrata , can be maintained indefinitely, free of microorganisms, on a diet of crystalline cellulose. Under these conditions the RQ is 1, indicating that the cockroach is surviving on glucose produced by endogenous cellulase. 3. 3. The in vitro rate at which glucose is produced from crystalline cellulose by gut extracts from P. cribrata and Nasutitermes walkeri is comparable to the in vivo production of CO 2 in these insects, clearly indicating that the rate of glucose production from crystalline cellulose is sufficient for their needs. 4. 4. In all termites and cockroaches examined, cellulase activity was found in the salivary glands and predominantly in the foregut and midgut. These regions are the normal sites of secretion of digestive enzymes and are either devoid of microorganisms (salivary glands) or have very low numbers. 5. 5. Endogeneous cellulases from termites and cockroaches consist of multiple endo-β-1,4-glucanase (EC 3.2.1.4) and β-1,4-glucosidase (EC 3.2.1.21) components. There is no evidence that an exo-β-1,4-glucanase (cellobiohydrolase) (EC 3.2.1.91) is involved in, or needed for, the production of glucose from crystalline cellulose in termites or cockroaches as the endo-β-1,4-glucanase components are active against both crystalline cellulose and carboxymethylcellulose. 6. 6. There is no evidence that bacteria are involved in cellulose digestion in termites and cockroaches. The cellulase associated with the fungus garden of M. michaelseni is distinct from that in the midgut; there is little indication that the fungal enzymes are acquired or needed. Lower termites such as Coptotermes lacteus have Protozoa in their hindgut which produce a cellulase(s) quite distinct from that in the foregut and midgut.

METAZOAN CELLULASE GENES FROM TERMITES: INTRON/EXON STRUCTURES AND SITES OF EXPRESSION

Endogenous endo-beta-1,4-glucanase (EGase, EC 3.2.1.4) cDNAs were cloned from representatives of the termite families Termitidae and Rhinotermitidae. These EGases are all composed of 448 amino acids and belong to glycosyl hydrolase family 9 (GHF9), sharing high levels of identity (40-52%) with selected bacterial, mycetozoan and plant EGases. Like most plant EGases, they consist of a single catalytic domain, lacking the ancillary domains found in most microbial cellulases. Using a PCR-based strategy, the entire sequence of the coding region of NtEG, a gene putatively encoding an EGase from Nasutitermes takasagoensis (Termitidae), was determined. NtEG consists of 10 exons interrupted by 9 introns and contains typical eukaryotic promoter elements. Genomic fragments of EGase genes from Reticulitermes speratus (Rhinotermitidae) were also sequenced. In situ hybridization of N. takasagoensis guts with an antisense NtEG RNA probe demonstrated that expression occurs in the midgut, which contrasts to EGase expression being detected only in the salivary glands of R. speratus. NtEG, when expressed in Escherichia coli, was shown to have in vitro activity against carboxymethylcellulose.

Cellulose and Xylan Utilisation in the Lower Termite Reticulitermes speratus

The distribution of the enzymes of cellulose and xylan metabolism namely endo-beta-1,4-glucanase, beta-glucosidase, endo-beta-1,4-xylanase and beta-xylosidase activities, in Reticulitermes speratus (Kolbe) was measured both in the salivary glands and in the major gut sections and along the length of the gut in freshly collected termites. The majority of the endo-beta-1,4-glucanase activity (77.8%) was found in the salivary glands which also contained 23.9% of the beta-glucosidase activity. At least 70% of the remaining activity was located in the anterior section of the hindgut. A small amount of endo-beta-1,4-xylanase activity (2.4%), but no beta-xylosidase activity, was present in the salivary glands. The majority of these activities were in the anterior section of the hindgut. The RQ of freshly collected termites at 25 degrees C was 1.03+/-0.01. Maintaining termites for 16 days on wood, cellulose and xylan showed that the RQ values of termites fed on wood or xylan were not significantly different from those of freshly collected termites but significantly increased when maintained on cellulose. The RQ of starved termites after 11 days was 0.81+/-0.02. There were three effects on protozoan populations of feeding termites xylan for 20 days. One species, Dinenympha parva was not affected, while five others, Pyrsonympha grandis, Holomastigotes elongatum, Dinenympha rugosa, Dinenympha leidy and Dinenympha porteri survived for 20 days but slowly decreased in numbers. The numbers of P. grandis and D. leidy surviving for 20 days were significantly different from those in starved termites. The third group comprising the two large species, Teratonympha mirabilis and Trichonympha agilis and three small species, Pyrsonympha modesta, Dinenympha exilis and Dinenympha nobilis disappeared within 15 days as in starved termites. It is suggested that protozoa in the first two groups are xylanolytic. Protozoan populations on wood and cellulose diets were not markedly affected. Selective removal of the protozoa by u.v. irradiation led to the loss of xylanolytic activity and a life span comparable to starved termites. Copyright 1997 Elsevier Science Ltd. All rights reserved

Dependence of the higher termite, Nasutitermes exitiosus and the lower termite, Coptotermes lacteus on their gut flora

The importance of the gut microorganisms in the termites Nasutitermes exitiosus and Coptotermes lacteus was investigated by feeding them with antibiotics. With N. exitiosus, antibiotics which killed both the bacteria and the spirochaetes (ampicillin, kanamycin, chloramphenicol, erythromycin, cephaloridine, tetracycline) reduced the life span of the termite from 250 days to about 13 days, whereas antibiotics which had little effect on the flora (penicillin, methicillin) did not greatly reduce the life span of the termite. The essential role of the spirochaetes in N. exitiosus was shown by feeding metronidazole, or exposing the termites to pure oxygen. Both treatments killed the spirochaetes, but not the bacteria, resulting in a life span for the termite of 13–22 days. Acid fuchsin did not kill the spirochaetes. Fungi were not essential for N. exitiosus. In C. lacteus all treatments, except that with acid fuchsin, killed the protozoa, thereby reducing the life span of the termite from 69 days to 6–29 days.

Digestion, diet and polyethism in two fungus-growing termites: Macrotermes subhyalinus Rambur and M. michaelseni Sjostedt

Abstract Cellulase and its component activities, endo-β-1,4-glucanase and β-1,4-glucosidase, were measured in the guts of larvae, minor soldiers and the four categories of workers (young and old minor and young and old major) in Macrotermes michaelseni and M. subhyalinus . These activities were also measured in fungal material associated with colonies of both species. Substantial activity cellulase (18.2–28.1 μg glucose/termite/h) and its component activities were found in all four worker categories of M. michaelseni with 80% less activity in the minor soldiers and none in the larvae. There was a similar distribution of cellulase activity among the castes of M. subhyalinus . At least 90% of the cellulase activity in major workers of M. michaelseni was found in the midgut. The only fungal material associated with either species with significant enzyme activity were the fungal nodules (cellulase activity, 8.22 ± 0.86 and 4.34 ± 0.24 μg glucose/mg wet wt for M. michaelseni and M. subhyalinus , respectively). The cellulases from major workers and fungal nodules from M. michaelseni were partially resolved by gel chromatography. Endo-β-1,4-glucanases and β-1,4-glucosidases from both cellulases were multi-component with different elution profiles on Bio-Gel ® P60 chromatography. Glucose and cellobiose were present throughout the gut of M. michaelseni . No other reducing sugars were present. Reducing sugars were found in all fungal material associated with M. michaelseni . In the nodules the only reducing sugar was glucose (184 ± 83 nmol/mg dry wt). Old fungal comb contained glucose, cellobiose, xylose, arabinose and galacturonic acid (13 ± 3, 5 ± 3, 15 ± 8, 24 ± 3, 12 ± 3 nmol/mg dry wt, respectively). Fresh and ripe fungal comb contained smaller amounts of the same sugars. The RQ values for young and old major workers of M. michaelseni were 0.999 ± 0.007 (SD; n = 4) and 1.002 ± 0.005 (SD; n = 5), respectively. Oxygen was consumed by the two categories at rates of 180 and 230 nmol/termite/h.

The site of cellulose breakdown in higher termites (Nasutitermes walkeri and Nasutitermes exitiosus)

Abstract Cellulase activity in the higher termite Nasutitermes walkeri is secreted predominantly in the midgut and to a minor extent in the salivary glands. 90% or more of each of the three compounds of cellulase activity, exo-β-1,4-glucosidase, β-1,4-glucosidase and endo-β-1,4-glucanase is found in the midgut. β-1,4-Glucosidase and endo-β-1,4-glucanase are confined to the anterior portion of the midgut. These activities increase to maximal values about one third the length of the midgut and then decrease symmetrically, presumably by resorption. Within the epithelial cells of this region β-1,4-glucosidase activity is mostly associated with the plasmalemma, though it is generally diffused through the cell. About 70% of β-1,4-glucosidase and endo-β-1,4-glucanase activities in the midgut are found in the lumen. Traces of all three components of activity are erratically distributed in the paunch. There is no bacterial cellulase activity in the paunch in either N. walkeri or N. exitiosus though the major carbohydrate components of wood are present in significant quantities in each section of the gut in N. exitiosus . Low levels of amylase activity are present in N. walkeri . It is secreted in the salivary glands (18%) and the midgut (73%) with traces found in the other gut sections. In the midgut it is only found in the anterior portion of the midgut.

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

Abstract 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.

Morphological, microbiological and biochemical studies of the gut flora in the fungus-growing termite Macrotermes subhyalinus

The four worker castes of the fungus-growing higher termite Macrotermes subhyalinus (Termitidae, Macrotermitinae) were examined to compare the morphology, physiology and microbiology of the alimentary canal. In young major and old major worker castes the midgut made up 64.8% of total gut volume, while in young minor and old minor workers the figure was significantly less at 53.0% (P < 0.05). Despite this morphological difference, all four castes supported a heterogeneous bacterial flora throughout the gut, with the highest densities (up to 1.3 x 1011 organisms ml−1) in the paunch and colon. Enumerations of the paunch, colon and rectum suggested an increase as both major and minor workers age. Intestinal pH was close to neutrality in the crop, midgut, colon and rectum, but alkaline in the paunch (pH 9.5 in young major and young minor workers). Redox indicator dyes fed to young major and young minor workers suggested that mildly reducing conditions existed in the paunch, colon and rectum (Eh = − 123 mV). Acetate concentration in whole gut homogenates of freshly sampled termites ranged from 6.39 mM in young majors to 18.26 mM in old minors (based on whole gut volume). Other short chain fatty acids, notably isovalerate, were present in small amounts. Hydrogen and methane were emitted by all four castes but molar rates were, respectively, no more than 1.55 and 1.51% that of CO2 production in any single caste, averaging 0.79% (H2) and 0.71% (CH4) over all castes. Weight specific methane emissions ranged from 0.016 nmolh mg in young and old minor workers to 0.141 nmolh mg in old major workers. These descriptive data on the gut flora of Macrotermes are broadly similar to those reported for members of other subfamilies of higher termites.

Symbiont-independent digestion of cellulose and starch in Panesthia cribrata Saussure, an Australian wood-eating cockroach

Abstract Respiratory quotient values (i.e. carbon dioxide produced/oxygen consumed) of 0.98–1.03 were obtained when Panesthia cribrata was maintained on diets of wood, Avicel, filter paper or starch; this is consistent with only carbohydrate being metabolized for energy production by the cockroach. The rate of carbon dioxide production on various cellulose and starch diets at 22°C (2.9–6.9 μmol/h/g cockroach) indicates that glucose is being metabolised in vivo at 0.46–1.15 μmol/h/g cockroach. The in vitro rate of glucose production from crystalline cellulose in whole gut homogenates is 1.05 ± 0.09 μmol/h/g cockroach. Endogenous cellulase is secreted in the epithelium of the anterior ventriculus and is found predominantly (98%) in the foregut and the anterior ventriculus. The cellulase consists of two main components, an endo-β-1,4-glucanase (EC 3.2.1.4) Mr, 2.4 × 104) which is active predominantly against carboxymethyl cellulose and a β-1,4-glucosidase (EC 3.2.1.21) (Mr, 1.2 × 105) which is active predominantly against cellobiose. The β-1,4-glucosidase activity is inhibited by glucono-δ-lactone and is thus true cellobiase activity. The cockroaches are not dependent on protozoa for survival or for cellulase as cockroaches maintained on tetracycline-impregnated filter paper show no change in viability or in cellulase activity. α-Amylase (EC 3.2.1.1) (Mr 5.9 × 104) activity is distributed in the gut in a similar way to cellulase activity and cockroaches can be maintained on starch for at least 12 weeks.