Srinivas Kambhampati

Nitrogen recycling and nutritional provisioning by Blattabacterium, the cockroach endosymbiont

Nitrogen acquisition and assimilation is a primary concern of insects feeding on diets largely composed of plant material. Reclaiming nitrogen from waste products provides a rich reserve for this limited resource, provided that recycling mechanisms are in place. Cockroaches, unlike most terrestrial insects, excrete waste nitrogen within their fat bodies as uric acids, postulated to be a supplement when dietary nitrogen is limited. The fat bodies of most cockroaches are inhabited by Blattabacterium, which are vertically transmitted, Gram-negative bacteria that have been hypothesized to participate in uric acid degradation, nitrogen assimilation, and nutrient provisioning. We have sequenced completely the Blattabacterium genome from Periplaneta americana. Genomic analysis confirms that Blattabacterium is a member of the Flavobacteriales (Bacteroidetes), with its closest known relative being the endosymbiont Sulcia muelleri, which is found in many sap-feeding insects. Metabolic reconstruction indicates that it lacks recognizable uricolytic enzymes, but it can recycle nitrogen from urea and ammonia, which are uric acid degradation products, into glutamate, using urease and glutamate dehydrogenase. Subsequently, Blattabacterium can produce all of the essential amino acids, various vitamins, and other required compounds from a limited palette of metabolic substrates. The ancient association with Blattabacterium has allowed cockroaches to subsist successfully on nitrogen-poor diets and to exploit nitrogenous wastes, capabilities that are critical to the ecological range and global distribution of cockroach species.

Taxonomy and Phylogeny of Termites

Termites are a large and diverse group, comprising roughly 281 genera and over 2600 described species. At present, seven families and 14 subfamilies are recognized. A large majority (ca. 85%) of known genera are included in one family, Termitidae. The present classification, as used since 1972, has been moderately stable but suffers from a lack of good defining synapomorphies for several groups and a rearrangement of taxa is desirable. At the evolutionary level, phylogenetic hypotheses proposed for Isoptera have one or more of these failings: a lack of cladistic analysis, too few characters, or poor taxon sampling. Surprisingly, the first cladistic phylogenetic hypothesis was published only in 1996, but three further studies have been overtaken subsequently. Mastotermitidae are generally accepted as the basal family, but in contrast there is little consensus on relationships among the sub-basal families and among subfamilies within the Termitidae. Recent advances in molecular techniques, cladistic analyses and increased cooperation among scientists employing disparate character sets presage a resolution of these problems. Appendices list described genera and estimate species numbers. There is an annotated key to families and subfamilies.

Developmental Origin and Evolution of Bacteriocytes in the Aphid–Buchnera Symbiosis

Symbiotic relationships between bacteria and insect hosts are common. Although the bacterial endosymbionts have been subjected to intense investigation, little is known of the host cells in which they reside, the bacteriocytes. We have studied the development and evolution of aphid bacteriocytes, the host cells that contain the endosymbiotic bacteria Buchnera aphidicola. We show that bacteriocytes of Acyrthosiphon pisum express several gene products (or their paralogues): Distal-less, Ultrabithorax/Abdominal-A, and Engrailed. Using these markers, we find that a subpopulation of the bacteriocytes is specified prior to the transmission of maternal bacteria to the embryo. In addition, we discovered that a second population of cells is recruited to the bacteriocyte fate later in development. We experimentally demonstrate that bacteriocyte induction and proliferation occur independently of B. aphidicola. Major features of bacteriocyte development, including the two-step recruitment of bacteriocytes, have been conserved in aphids for 80–150 million years. Furthermore, we have investigated two cases of evolutionary loss of bacterial symbionts: in one case, where novel extracellular, eukaryotic symbionts replaced the bacteria, the bacteriocyte is maintained; in another case, where symbionts are absent, the bacteriocytes are initiated but not maintained. The bacteriocyte represents an evolutionarily novel cell fate, which is developmentally determined independently of the bacteria. Three of five transcription factors we examined show novel expression patterns in bacteriocytes, suggesting that bacteriocytes may have evolved to express many additional transcription factors. The evolutionary transition to a symbiosis in which bacteria and an aphid cell form a functional unit, similar to the origin of plastids, has apparently involved extensive molecular adaptations on the part of the host cell.

Molecular cloning and characterization of a greenbug (Schizaphis graminum) cDNA encoding acetylcholinesterase possibly evolved from a duplicate gene lineage.

An acetylcholinesterase (AChE, EC 3.1.1.7) cDNA was cloned and characterized from a greenbug (Schizaphis graminum (Rondani)) cDNA library. The complete cDNA (3283 bp) contains a 2028-bp open reading frame encoding 676 amino acid residues. The putative AChE preproenzyme has a 17 amino acid signal peptide, a 78 amino acid activation peptide and a mature enzyme of 581 amino acid residues. The first nine amino acid residues (YTSDDPLII) that were determined by sequencing the N-terminus of a 72-kDa AChE purified from the greenbug matched the nine residues deduced from the cDNA. The key amino acid residues, including the three residues Ser206 (200 in Torpedo), Glu332 (327) and His446 (440) forming a catalytic triad, three pairs of cysteine putatively forming intrachain disulfide bonds, and 10 out of the 14 aromatic residues lining the active site gorge of the Torpedo AChE, are conserved. However, Ser336 (Phe331) in the greenbug substituted an aromatic amino acid residue that is conserved in all other known AChEs. Northern blot analysis of mRNA revealed a 3.7-kb transcript, and Southern blot analysis suggested a single copy of this gene in the greenbug. The deduced amino acid sequence is most similar to AChE1 of the nematodes Caenorhabditis briggsae and C. elegans with 43% identity. Phylogenetic analysis showed that the greenbug AChE formed a cluster with those of nematodes, a squid and ticks, and grouped out of the insect cluster. This result suggests that the cloned gene evolved from a different duplicate gene lineage of insect AChEs.

Phylogenetic relationship among termite families based on DNA sequence of mitochondrial 16S ribosomal RNA gene.

Termites (Order isoptera: Class Insecta), are comprised of a complex assemblage of species, with considerable variation in life history, morphology, social behaviour, caste development and ecology. At present, isoptera is divided into seven families, fourteen subfamilies, ∼ 270 genera and over 2000 species. Phylogenetic hypotheses currently available for termite families and genera are based on a limited number of morphological characters and lack rigorous cladistic analysis. In this paper we report on phylogenetic relationships among ten termite genera of five families based on a DNA sequence analysis of a portion of the mitochondrial 16S rRNA gene. Parsimony and distance analysis of DNA sequences supported the existing hypothesis that Mastotermitidae is the basal lineage among extant termites. Kalotermitidae was not found to be a sister taxon of Mastotermitidae as existing hypotheses suggest, but was most closely related to Rhinotermitidae and Termitidae. Representatives of Termopsidae were more basal relative to those of Kaiotermitidae. The utility of 16S rRNA nucieotide sequence analysis for inferring phylogenetic relationships among termite families, subfamilies and genera is discussed.

Coevolution between a cockroach and its bacterial endosymbiont: a biogeographical perspective

Cryptocercus are subsocial, xylophagous cockroaches that live in temperate forests. Like other cockroaches, Cryptocercus harbour endosymbiotic bacteria in their fat bodies. Two species of Cryptocercus occur in the palaearctic, one each in eastern Russia and south-central China. In the USA, there are five species: one in the north-west and four in the south-east. Little is known about the relationship between the Eurasian and North American Cryptocercus or the causes of the disjunct distribution. Here, a molecular phylogeny for six out of the seven Cryptocercus species and their endosymbionts is inferred in an attempt to understand the evolution and biogeography of the genus. Our analysis showed that the North American Cryptocercus are monophyletic, suggesting that a single colonization event was followed by vicariance. There was complete concordance between the host and endosymbiont phylogenetic trees. Divergence estimates based on endosymbiont DNA sequences suggested that the palaearctic and nearctic Cryptocercus diverged 70–115 million years (Myr) ago and the eastern- and western-USA species diverged 53–88 Myr ago. These divergence estimates were correlated with biogeographical events, and a hypothesis is presented to explain the current distribution of Cryptocercus. Our findings suggest that Cryptocercus has had a long evolutionary history, dating back to the Jurassic.

Phylogenetic relationships among genera of Aphidiinae (Hymenoptera : Braconidae) based on DNA sequence of the mitochondrial 16S rRNA gene

Phylogenetic relationships among forty‐nine taxa representing twenty‐four genera of Aphidiinae (Hymenoptera: Braconidae) were investigated using DNA sequence of a portion of the mitochondrial 16S rRNA gene and parsimony analysis. Seven species in six other subfamilies of Braconidae were used as outgroup. The results suggested that members of Aphidiinae are monophyletic. The basal lineage of Aphidiinae was Aclitus in weighted and unweighted parsimony analyses and Praini was basal relative to Ephedrini. With the exception of Pauesia and Aphidius, all genera were monophyletic. The results support generic status for Euaphidius, but not for Lysaphidus. Diaeretus leucopterus was internal to a clade composed of three Pauesia species, suggesting that the latter genus may be paraphyletic. A combined analysis that included DNA sequence of 16S rRNA, NADH1 dehydrogenase and 28S rRNA resulted in more robust cladograms with topologies similar to those inferred from the 16S rRNA gene sequence alone. The results are compared to previously proposed phylogenies of Aphidiinae based on morphological and molecular characters.

Phylogenetic relationship among cockroach families inferred from mitochondrial12S rRNA gene sequence

A number of phylogenies exist for cockroaches that differ in the postulated relationships among families and genera. The relationship of the wood‐feeding genus, Cryptocercus, to other cockroach families and to termites, has generated considerable debate. Grandcolas (1994), based on morphological analysis, synonymized the family Cryptocercidae with Polyphagidae and placed the genus Cryptocercus in the subfamily Polyphaginae. To determine if an independent set of characters supports the placement of Cryptocercus in Polyphaginae, a phylogenetic analysis of relationships among representative genera of the five cockroach families was undertaken. DNA sequence of a ‐430 base pair portion of the mitochondrial small ribosomal subunit gene from representatives of Blattidae, Blattellidae, Blaberidae and Cryptocercus, previously published by Kambhampati (1995) and Kambhampati et al. (1996), and the homologous sequence from representatives of Polyphagidae were used in the analysis. A total of twenty cockroach taxa and three termite genera were included in the study. Because a recent study showed that Cryptocercus punctulatus consists of a species complex, DNA sequence from four individuals collected in different parts of the U.S.A. was included in the study. The trees estimated from parsimony and neighbour‐joining analyses indicated that Cryptocercus is a monophyletic clade which is most closely related to members of Blattidae. Polyphagidae is indicated as a sister group to the Blattidae + Cryptocercus complex, suggesting that Polyphagidae may belong to the superfamily Blattoidea rather than to Blaberoidea as proposed by McKittrick (1964). Blaberidae and Blattellidae were sister groups as previously proposed. Based on the present analysis, I propose that the genus Cryptocercus be retained in the family Cryptocercidae. Cockroaches

Biochemical mechanisms of organophosphate resistance in Rhyzopertha dominica (Coleoptera: Bostrichidae) populations from the United States and Brazil

Four possible biochemical mechanisms of organophosphate resistance were examined in populations of Rhyzopertha dominica (Fabricius) collected from eight locations in the states of Minas Gerais and Sao Paulo in Brazil and from seven locations in northeast Kansas, United States. Results from insecticide bioassays with synergists (triphenyl phosphate, diethylmaleate, and piperonyl butoxide) suggested that metabolic detoxification mechanisms might be involved in organophosphate resistance in some of the populations of R. dominica . However, overall low synergism, non-synergism or even antagonism presented by these synergists in all 15 resistant populations suggested that carboxylesterases, cytochrome P450 monooxygenases and glutathione S -transferases were not likely to play a major role conferring organophosphate resistance in R. dominica . This hypothesis was strongly supported by our biochemical studies showing no significant difference in these enzyme activities, as well as cytochrome b 5 , P420, and P450 levels between the susceptible and resistant populations. In contrast, all resistant populations of R. dominica showed higher acetylcholinesterase activity than the susceptible population both in the presence and absence of malaoxon. Furthermore, acetylcholinesterase from the resistant populations was less sensitive to inhibition by malaoxon than that from the susceptible. These results suggested that quantitative and qualitative changes in acetylcholinesterase might contribute to organophosphate resistance in these populations of R. dominica .

Evidence for sibling species in Cryptocercus punctulatus, the wood roach, from variation in mitochondrial DNA and karyotype.

The wood-feeding genus Cryptocercus is considered the basal lineage among extant cockroaches. Cryptocercus is the sole representative of the family Cryptocercidae and at present three species are recognized within the genus worldwide: Cryptocercus punctulatus in the United States, C. relictus in Eurasia and C. primarius in the Orient. The geographical distribution of C. punctulatus in the USA is disjunct, with populations occurring along the Appalachian Mountains and in the Pacific North-west. In samples collected from several locations of the eastern and the western USA, we investigated variation in DNA sequence of portions of the two mitochondrial rRNA genes and in chromosome number. The overall sequence divergence among 30 individuals assayed from 17 locations was 17.3 per cent. A phylogenetic analysis revealed that in the east, individuals in Virginia had diverged significantly in their haplotype from those in North Carolina, Georgia and Alabama; individuals in the west (Oregon) had diverged in their haplotype from individuals in the east. The diploid chromosome number for 52 male C. punctulatus sampled from 15 locations varied from 37 (18II + X) to 47 (23II + X). In the eastern samples, the diploid chromosome number ranged from 37 to 45, whereas in Oregon all individuals had 2n = 47. No polymorphism in DNA sequence or chromosome number among individuals collected within a locality was detected. The DNA sequence and chromosome number variation data, together with preliminary studies on mating incompatibility, strongly suggest that C. punctulatus in the USA is comprised of at least two and probably three sibling species, with one species occurring in western USA and one or more species in eastern USA.