The Cryptocercus punctulatus species complex (Dictyoptera: Cryptocercidae) in the eastern United States: comparison of cuticular hydrocarbons, chromosome number, and DNA sequences.

The goal of the current study was to determine if cuticular hydrocarbons could be used to empirically delimit taxa within the Cryptocercus punctulatus species complex in the eastern United States. Cockroaches were collected from rotting logs in 22 locations across four states. Hydrocarbon phenotypes and two mitochondrial (16S and COII) genes and one nuclear (ITS2) gene were independently analyzed to determine their relationship with chromosome number. Five distinct hydrocarbon phenotypes were found, but these were only partly congruent with chromosome number and thus with purported species descriptions. Molecular and cuticular hydrocarbon data each indicate that Cryptocercus with a male karyotype of 2n=43 belong to at least two distinct, distantly related lineages. One 2n=43 lineage is sister group to the 2n=37 and 2n=39 clade, and has a unique hydrocarbon profile. The other 2n=43 lineage is sister group to the 2n=45 samples, and its cuticular hydrocarbons group with four samples of the 2n=45 lineage. The cuticular hydrocarbons of two additional 2n=45 samples diverge from this assemblage. Results indicate cuticular hydrocarbons and chromosome number have some degree of evolutionary independence; neither is completely reliable in delineating historical lineages. Our data provide support for the parallel model of chromosome evolution in the species complex.

The ootheca of Mastotermes darwiniensis Froggatt (Isoptera: Mastotermitidae): homology with cockroach oothecae.

The basal termite Mastotermes darwiniensis produces an egg mass, the nature of which is controversial. The debate centres on whether it is homologous with the oothecae of mantids and cockroaches and, if so, whether its simple structure is plesiomorphic or apomorphic within the Dictyoptera. To help resolve these issues we observed primary reproductives of M. darwiniensis during oviposition and examined the morphology of the reproductive product. Oviposition is cockroach-like in that the egg mass is assembled within the vestibulum and the eggs are issued externally in pairs. The reproductive product is an ootheca of the blattarian type. A distinct, tanned outer covering is stretched over the two parallel rows of eggs. No keel is present and no calcium oxalate crystals were apparent in the outer covering. We cannot rule out the possibility that the simple structure of the ootheca is plesiomorphic within Dictyoptera. However, based on (i) apomorphies shared by Mastotermes and Blattaria, and (ii) the life habits of Isoptera, a secondary reduction is the more plausible explanation.

Some aspects of intracellular symbiosis during embryo development of Mastotermes darwiniensis (Isoptera: Mastotermitidae).

All examined species of cockroaches have been shown to harbour intracellular bacteria in specialized cells (bacteriocytes) of the fat body. In termites, bacteria in specialized cells have been observed only in Mastotermes darwiniensis (Isoptera: Mastotermitidae). All of these bacteria have been assigned to the same eubacterial lineage, with the bacteria of M. darwiniensis as the sister group to the cockroach bacteria. While the main steps of the life cycle of cockroach bacteria have been described, little is known about the bacteria of M. darwiniensis. More specifically, no data are available on their behaviour during the development of this termite. Using both optical and electron microscopy methods, we examined embryos of M. darwiniensis at different developmental stages. Our results show that the integration of bacteria during the development of M. darwiniensis is implemented in the same way as in cockroaches. In particular, we observed the aggregation of a large amount of bacteria in a single mass in the yolk sac, with vitellophage-associated bacterial lysis. In cockroaches, a similar process has been described in detail for Periplaneta americana (Blattaria: Blattidae), where the bacterial mass is referred to as the transitory mycetome. The formation of a transitory mycetome could thus be regarded as an ancestral condition for cockroaches and termites.

Phylogenetically distant intracellular symbionts in termites.

Cockroaches are known to harbour intracellular bacteria in specialised cells (mycetocytes, or bacteriocytes) of the fat body. In termites, mycetocyte bacteria have been observed only in Mastotermes darwiniensis. These symbionts are thought to have originated from a bacterium that infected an ancestor common to cockroaches and termites. Thus, loss of the infection should have occurred during evolution in all termite lineages, with the exception of that leading to M. darwiniensis. One might suspect that traces of the ancient infection may be present in some termites, in the form of non-mycetocyte intracellular bacteria (e.g. a small number of bacteria within normal cells). Indeed, circumstantial evidence for the presence of intracellular bacteria in two termite species has been reported. However, no data are available on the actual distribution of these bacteria in termites, or on their relationships with the mycetocyte bacteria of cockroaches and M. darwiniensis. In this paper we report results indicating that non-mycetocyte intracellular bacteria are widespread in termites. These results were obtained by electron microscopy on representatives of nine termite species. In addition, sequence analysis of the 16S rRNA genes indicated that the non-mycetocyte bacteria of termites belong to the wolbachia group of the alpha-2 subclass of the proteobacteria. These latter bacteria are not related to the mycetocyte bacteria of cockroaches and M. darwiniensis, which belong to the blattabacterium group of the flavobacteria-bacteroides. PCR analyses with primers specific for wolbachia or blattabacterium provided further support for the identification of the observed non-mycetocyte bacteria as members of the wolbachia group.

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 geological 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 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 percent. 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 (18(II) + X) to 47 (23(II) + 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 probably three sibling species, with one species occurring in western USA and one or more species in eastern USA.

The establishment of intracellular symbiosis in an ancestor of cockroaches and termites.

All cockroaches examined so far have been found to harbour a bacterial endosymbiont in specialized cells of the fat body, whereas Mastotermes darwiniensis is the only termite currently known to harbour an intracellular symbiont. The localization and mode of transmission of these bacteria are surprisingly similar, but so far no data have been published on their phylogenetic relationships. To address this issue, molecular sequence data were obtained from the genes encoding the small subunit ribosomal RNA of the M. darwiniensis endosymbiont, and compared with those obtained from endosymbionts of seven species of cockroaches. Molecular phylogenetic analysis unambiguously placed all these bacteria among the flavobacteria-bacteroides, indicating that the endosymbiont of M. darwiniensis is the sister group to the cockroach endosymbionts examined. Additionally, nucleotide divergence between the endosymbionts appears to be congruent with the palaeontological data on the hosts's evolution. These results support previous claims that the original infection occurred in an ancestor common to cockroaches and termites. A loss of endosymbionts should subsequently have occurred in all termite lineages, except that which gave rise to M. darwiniensis.

Bird dispersal of the larval stage of a seed predator.

Multiflora rose seeds containing larvae of the phytophagous wasp Megastigmus aculeatus nigroflavus were fed to a caged mockingbird (Mimus polyglottos). Ninety percent of the seeds were recovered from the bird's feces, and there was no significant difference in subsequent emergence of the wasps from these seeds when compared to controls. This study indicates the dispersal of seed-feeding hymenoptera by avian frugivores may be a widespread and significant phenomenon.

Ancestral transfer of symbionts between cockroaches and termites: an unlikely scenario.

Thorne's (Proc. R. Soc. Lond. B 241, 37 (1990] studies of the laboratory behaviour of extant woodroaches (Cryptocercus) and termites (Zootermopsis) suggest that transfaunation of hindgut protozoans potentially could occur by aggression and consumption in the field. However, existing literature suggests no overlap in protozoan species composition for these two taxa. Furthermore, it is doubtful that transfaunation would occur in the solitary ancestral 'termitoid' and 'roachoid' lines proposed by Thorne: not only is it unlikely that such insects would encounter each other, but it is doubtful that they would show the degree of aggression exhibited by the termite soldiers in her study. Inheritance from an ancestor common to Cryptocercus and the lower termites remains the most logical explanation for the presence of the unique cellulolytic oxymonad, trichomonad and hypermastigote flagellates in these two groups.

Isolation and identification of 4,6,8-trimethyl-7,9-undecadien-5-ol, a female-specific compound, in tergal gland secretion ofCryptocercus punctulatus scudder (Dictyoptera: Cryptocercidae).

The secretion of the tergal glands ofCryptocercus punctulatus consists of a complex mixture of 27 compounds. Of this mixture, only one compound (compound B) is specific for females. By dissecting 200 tergal glands, 50 µg of pure compound B was collected by preparative GC; it was identified as (Z, E-4,6,8-trimethyl-7,9-undecadien-5-oI. Its functions as well as its absolute configuration still have to be determined.

Tergal glands of male and femaleCryptocercus punctulatus scudder (Dictyoptera: Cryptocercidae): Composition, sexual dimorphism, and geographic variation of secretion.

Males and females ofCryptocercus punctulatus possess tergal glands, but they differ in position, size, morphology, and secretion chemistry. Compound A (linalyl acetate) is the most abundant of the 21 compounds found only in the secretion of these glands. Compound B, 4,6,8-trimethyl-7,9-undecadien-5-ol, is specific to the tergal secretion of females.C. punctulatus lives only in the United States; its distribution is disjunct. Compound A is found in samples from the eastern population but is absent in samples from the western population. The amount of compound B per gland in samples from the western population is at least twice as high as in the samples from the eastern populations.