Harley A. Rose

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.

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 June 2010 - 31 July 2010.

This article documents the addition of 205 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Bagassa guianensis, Bulweria bulwerii, Camelus bactrianus, Chaenogobius annularis, Creontiades dilutus, Diachasmimorpha tryoni, Dioscorea alata, Euhrychiopsis lecontei, Gmelina arborea, Haliotis discus hannai, Hirtella physophora, Melanaphis sacchari, Munida isos, Thaumastocoris peregrinus and Tuberolachnus salignus. These loci were cross‐tested on the following species: Halobaena caerulea, Procellaria aequinoctialis, Oceanodroma monteiroi, Camelus ferus, Creontiades pacificus, Dioscorea rotundata, Dioscorea praehensilis, Dioscorea abyssinica, Dioscorea nummularia, Dioscorea transversa, Dioscorea esculenta, Dioscorea pentaphylla, Dioscorea trifida, Hirtella bicornis, Hirtella glandulosa, Licania alba, Licania canescens, Licania membranaceae, Couepia guianensis and 7 undescribed Thaumastocoris species.

Increased glutathione S-transferase activity and glutathione content in an insecticide-resistant strain of Tribolium castaneum (Herbst)

Abstract Cyfluthrin resistance in Tribolium castaneum (Herbst) was associated with increases in both glutathione (GSH) concentration and glutathione S-transferase (GST) activity. Cytosolic GSH content was increased twofold in resistant beetles. Similarly, conjugation of the GST substrates 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB) were elevated four- to sixfold in cytosols isolated from resistant beetles. However, no difference in GST activity was noted when p-nitrobenzylchloride (PNBC) was used as a substrate. Kinetic analysis of cytosolic CDNB activity revealed a four- to fivefold increase in apparent Vmax in resistant cytosols. Apparent Km, however, was similar in both resistant and susceptible cytosols. Partial purification of beetle cytosols revealed increased CDNB activity in resistant cytosols eluting as the unbound fraction following DE-52 chromatography when compared with cytosols isolated from susceptible insects. When electrophoresed on an equal protein basis, SDS-PAGE analysis revealed increased staining of a single protein band in the GST molecular weight region (24–30,000 Da) in cytosols isolated from resistant beetles. Taken together, these results are consistent with the increased expression of a common GST in resistant beetles. Microsomes isolated from resistant beetles exhibited a type I binding spectrum when incubated with piperonyl butoxide. In contrast, no spectral interaction was noted when piperonyl butoxide was added to microsomes isolated from susceptible insects. Similarly, metabolic intermediate (MI) complex formation with piperonyl butoxide was only observed in microsomes from resistant beetles. Addition of GSH to the incubation mixture prior to the initiation of piperonyl butoxide metabolism resulted in a dose-related decrease in complex formation (as measured by 427 nm peak height), 5 mM GSH being associated with a 50% decrease in complexation. This result suggests that increased GSH levels may influence MI complex formation in vivo and hence affect insecticide synergism.

Glutathione S-transferase in the Australian sheep blowfly, Lucilia cuprina (Wiedemann)

Abstract Glutathione S -transferase in the Australian sheep blowfly, Lucilia cuprina , was studied using 3,4-dichloronitrobenzene (DCNB) and 1-chloro-2,4-dinitrobenzene (CDNB) as substrates. The optimum pHs for enzyme activity were 7.5–8.0 and 6.7–7.4 for DCNB and CDNB conjugations, respectively. Inclusion of glutathione and bovine serum albumin in the homogenizing buffer protected the glutathione S -transferase from inhibition by endogenous compounds present in extracts of final instar larvae and of adults less than 7–8 days old. Conjugation activities for DCNB and CDNB increased throughout larval development to reach a peak early in the pupal stage. Activity then decreased through the remainder of the pupal stage and for the first 6–7 days after emergence of the adult. Almost all of the decrease in activity during the first 6 days of the adult occurred in the abdomen, which accounted for 85% of total activity in the adult female at emergence but only 47% at 6 days. Larval DCNB conjugation activity was localized almost entirely in the fat body (94%), whereas only 50% of the CDNB conjugation activity was in the fat body with the remainder in the cuticle (25%), gut (15%), and blood (10%). Adult and larval enzyme was induced ca. three- to four-fold by sodium phenobarbital. The induction was associated with changes in apparent V max rather than apparent K m , suggesting that phenobarbital caused increased production of forms of enzymes already present rather than inducing synthesis of altered or new forms.

The evolution of soil-burrowing cockroaches (Blattaria: Blaberidae) from wood-burrowing ancestors following an invasion of the latter from Asia into Australia

Morphologically similar cockroaches in the subfamilies Panesthiinae and Geoscapheinae (Blattaria: Blaberidae) display contrasting feeding habits, behaviour and biogeographical distributions. Panesthiinae, found throughout Asia and Australia, all live in and feed on decaying wood that they burrow into. Geoscapheinae are restricted to Australia and construct and live in burrows in the soil, where they feed on dry leaves taken from the surface. A lack of knowledge about phylogenetic relationships among these cockroaches hinders an understanding of the factors that have shaped the evolution of their diverse lifestyles and biogeography. To address this issue, we sequenced three genes from representatives of nine of the 10 genera in the two subfamilies, and performed phylogenetic analyses. The well–supported topology revealed the Panesthiinae to be paraphyletic with respect to the Geoscapheinae. Soil–burrowing cockroaches appear to have evolved from a lineage of wood burrowers that invaded Australia from the north some time after the merging of the Asian and Australian tectonic plates ca. 20 Myr ago. The main factor promoting the evolution of soil burrowing is likely to have been one of the periods of strong aridity that Australia has experienced since the Miocene period.

Microsomal oxidase activity of three blowfly species and its induction by phenobarbital and β-naphtoflavone

Abstract Induction of the microsomal oxidase system by dietary phenobarbital and β-naphthoflavone was examined in three blowflies, Phormia regina (Mg.), Lucilia illustris (Mg.), and Eucalliphora lilica (Walk.). Responses were similar in adults and larvae of all species. Phenobarbital increased cytochrome P -450 levels up to 9-fold and aldrin epoxidase up to 138-fold. Increases in cytochrome P -450 and aldrin epoxidase caused by β-naphthoflavone were minor relative to those produced by phenobarbital. In toxicity experiments with carbaryl and propoxur tolerance was associated with the amount of microsomal oxidase activity. Using piperonyl butoxide to synergize carbaryl and propoxur there was no clear indication for the use of either the synergist ratio or synergist difference as an indicator of microsomal oxidase activity.

Evidence for cytochrome P-450 multiplicity in the midgut of the cluster caterpillar, Spodoptera litura (F.)

Abstract Treatment of Spodoptera litura (F.) (the cluster caterpillar) larvae with geraniol (0.4%), pentamethylbenzene (0.2%), β-naphthoflavone (0.2%), and phenobarbital (0.1%) increased cytochrome P -450 content, with geraniol causing a four-fold induction (no differential induction of cytochrome P -450 monooxygenase activity was observed). All treatments, except phenobarbital, increased aldrin epoxidase, ethoxyresorufin-, and ethoxycoumarin- O -deethylase activities. Only geraniol treatment induced epoxide hydrolase activity. Ethoxyresorufin and ethoxycoumarin- O -deethylase activities were insensitive to carbon monoxide and metyrapone inhibition. In contrast, aldrin epoxidase was sensitive to both inhibitors. Differential inhibition was also seen with α-naphthoflavone with the O -deethylase activities being more inhibited than aldrin epoxidase at equal inhibitor concentration. Subsequent studies with isosafrole (0.05%) resulted in qualitative changes in midgut microsomal cytochrome P -450 parameters compared to geraniol (0.4%) and control microsomes. Isosafrole treatment greatly induced ethoxyresorufin- (∼20-fold) and ethoxycoumarin- O -deethylase activity while only slightly inducing aldrin epoxidase activity (∼2-fold). Isosafrole treatments altered the apparent K m for ethoxyresourufin and ethoxycourmarin and increased the proportion of low-spin cytochrome P -450 present in midgut microsomes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of isosafrole-treated microsomes revealed the presence of two protein staining bands of molecular weights 53,500 and 50,500 not observed in control or geraniol-induced microsomes. The results suggest that like S. eridania multiple forms of cytochrome P -450 are present in S. litura midgut microsomes.

Effects of picloram on xenobiotic biotransformation in rat liver.

1. The effect of picloram on model xenobiotic substrate biotransformation in vivo was studied in female and male rat liver. 2. Treatment with picloram had little effect on epoxide hydratase and glutathione S-transferase activity, but caused a dose-dependent increase in ethoxyresorufin-O-deethylase activity and a concomitant decrease in aldrin epoxidase activity in male rats. 3. Treatment of male rats with equivalent doses of 2-acetylaminofluorene, 2-amino-anthracene and picloram induced ethoxyresorufin-O-deethylase activity to the same degree. 4. Treatment of female rats with picloram resulted in dose-dependent increases in ethoxyresorufin and ethoxycoumarin-O-deethylation without decreasing aldrin epoxidase activity. 5. Picloram binds to liver microsomal preparations from rats pretreated with phenobarbitone and/or 3-methylcholanthrene, giving a type I spectrum. 6. The results indicate that picloram is a 3-methylcholanthrene-type inducer, and the implications are discussed.

Effect of length of exposure to malathion on xenobiotic biotransformation in male rat liver

The effect of exposure to malathion on several parameters of hepatic xenobiotic biotransformation was studied in male Sprague-Dawley rats. Groups of rats dosed i.p. daily for 1 or 2 weeks with 40 or 200 mg/kg malathion showed an increase in epoxide hydrolase activity (1 week, 200 mg/kg) and glutathione S-transferase activity (1 week, 200 mg/kg; 2 weeks 40 and 200 mg/kg). Aldrin epoxidation was decreased after 1 week of exposure to 200 mg/kg and by both dosage regimens after 2 weeks. After 9 weeks exposure to 40 mg/kg malathion administered i.p. 3 times per week, however, no changes in hepatic xenobiotic biotransformation were noted. The results demonstrate that only continuous exposure to high doses of malathion results in an induction of epoxide hydrolase and glutathione S-transferase activities. Inductive effects on hepatic cytochrome P-450 monooxygenase activity were not observed irrespective of whether exposure was short- or medium-term.

Tyrosinase as an inhibitor of aldrin epoxidase in house fly microsomes

Abstract The alleged inhibition by the house fly tyrosinase system of microsomal aldrin epoxidase and its reversal by KCN, which has been reported by another laboratory, was investigated. It was found that, except in newly emerged adults or in late instar larvae, no such effect was evident. Although a full explanation of the reported low epoxidase activity was not possible, some contributing practices were identified. One of these was the use of highly concentrated homogenates in the preparation of house fly microsomes, resulting in low recoveries of enzyme activity. The stimulatory effect of KCN, added during homogenization, is attributed, at least in part, to its negative effect on the determination of microsomal protein, thus resulting in a positive error when epoxidase activity is based on microsomal protein. Tyrosinase is a potent inhibitor of aldrin epoxidase in the microsomes prepared from late instar house fly larvae and its effect can be greatly reduced by the use of KCN. Bovine serum albumin, added during homogenization of house fly larvae, also enhances the epoxidase. Together, KCN and BSA, can increase epoxidase activity sevenfold in such larvae.