Multiple evolutionary origins of Australian soil-burrowing cockroaches driven by climate change in the Neogene.

Parallel evolution is the independent appearance of similar derived phenotypes from similar ancestral forms. It is of key importance in the debate over whether evolution is stochastic and unpredictable, or subject to constraints that limit available phenotypic options. Nevertheless, its occurrence has rarely been demonstrated above the species level. Climate change on the Australian landmass over the last approximately 20 Myr has provided conditions conducive to parallel evolution, as taxa at the edges of shrinking mesic habitats adapted to drier biomes. Here, we investigate the phylogeny and evolution of Australian soil-burrowing and wood-feeding blaberid cockroaches. Soil burrowers (subfamily Geoscapheinae) are found in relatively dry sclerophyllous and scrubland habits, whereas wood feeders (subfamily Panesthiinae) are found in rainforest and wet sclerophyll. We sequenced and analysed mitochondrial and nuclear markers from 142 specimens, and estimated the evolutionary time scale of the two subfamilies. We found evidence for the parallel evolution of soil-burrowing taxa from wood-feeding ancestors on up to nine occasions. These transitions appear to have been driven by periods of aridification during the Miocene and Pliocene across eastern Australia. Our results provide an illuminating example of climate-driven parallel evolution among species.

Consideration for the validation of clinical laboratory methods in nonclinical fields.

In new drug development, cells or animals are treated with the selected candidate compound to confirm its efficacy and safety in nonclinical studies. Clinical laboratory tests are carried out using samples from experimental animals in these studies. The clinical laboratory test method validation in nonclinical fields should be conducted keeping in mind that the circumstances differ from those in clinical settings. However, the validation procedures have not been systematically integrated into any standard. The considerations in this paper set out systematically practical guidance for the validation of quantitative analytical methods for fluid samples collected from animal studies, for the purpose of ensuring that laboratory test method validation is conducted in nonclinical fields at an enough level.

The homolog of Ciboulot in the termite (Hodotermopsis sjostedti): a multimeric beta-thymosin involved in soldier-specific morphogenesis.

BACKGROUND: Caste differentiation in social insects is a type of polyphenism that enables division of labor among members of a colony. This elaborate social integration has attracted broad interest, although little is known about its regulatory mechanisms, especially in Isoptera (termites). In this study, we analyzed soldier differentiation in the damp-wood termite Hodotermopsis sjostedti, focusing on a possible effector gene for caste development. The gene for an actin-binding protein, HsjCib, which shows a high level of expression in developing mandibles during soldier differentiation, is characterized in detail. RESULTS: To examine the HsjCib gene, full-length cDNAs were obtained by rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR) and sequencing. Multiple isoforms were identified, and on the basis of the results of northern and Southern hybridization analyses, these isoforms were considered to be transcriptional variants from a single gene. On the basis of their sequence similarity to homologous genes of other organisms, functions in actin assembly were assumed to be different among isoforms. Expression analysis revealed high expression in the head during soldier differentiation, which was consistent with their allometric growth. Although isoform expression was observed in various tissues, different expression levels were observed among tissues, suggesting the possibility of tissue-specific morphogenetic regulation by HsjCib isoforms. CONCLUSION: This study revealed the characteristics and dynamics of the HsjCib gene during soldier differentiation as a potential representative of downstream effector genes in caste-specific morphogenesis. From the expression patterns observed, this gene is considered to be involved in cephalic morphogenesis and neural reorganization, resulting in the establishment of caste-specific morphology and behavior.

Altricial development in subsocial wood-feeding cockroaches.

Species in the wood-feeding genus Salganea within the cockroach subfamily Panesthiinae (Blaberidae) typically live in biparental families; their first instars suffer high mortality when removed from adults, and in at least one species, adults are known to feed neonates on oral liquids. In the closely related gregarious wood-feeding genus Panesthia , no parental interaction with offspring is known. We compared the external morphology of first instars of these two genera and found that eye development and cuticular pigmentation at hatching are correlated with social structure. First instars of Panesthia have a dark cuticle and well-developed eyes. First instars of Salganea have a pale, transparent cuticle, and eyes significantly smaller than those of Panesthia relative to adult eye size. The body size of the first-instar of Salganea esakii is significantly smaller than that of Panesthia angustipennis spadica , relative to adult body size (24.0 and 27.4% of adult head-capsule width, respectively), but a more general survey suggests that, overall, neonate size may be similar in the two genera. We compared the first instars of these two taxa of Panesthiinae to those in the biparental, wood-feeding cockroach Cryptocercus (Cryptocercidae) and discuss how decreased investment in both integumentary and ocular development in subsocial cockroaches parallels that seen in altricial vertebrates.

Relationship between dynamic change of copper and dioxin generation in various fly ash.

Only limited information on the chemical forms of copper in municipal solid waste incinerator (MSWI) fly ash is available in the literature. Therefore, we performed in situ X-ray absorption near-edge spectroscopy (XANES) experiments involving two types of real fly ash to confirm the behavior of copper in fly ash at secondary formation temperatures for polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs). CuCl(2).3Cu(OH)(2) was detected in both fly ash types before heating experiments. Cupric compounds in real fly ash were reduced to cuprous compounds or elemental copper at approximately 200 degrees C. The changes in the Cu XANES spectra observed for the two fly ash samples were similar to those observed previously. We also examined the behavior of copper in model fly ash using SiO(2) to investigate the effect of supporting materials and copper content in fly ash on the dynamic changes of copper and amount of dioxins generated. There was little difference between the use of boron nitride (BN) and SiO(2) as a supporting material. The formation mechanisms of PCDDs and PCDFs in both of the model fly ash types were basically the same. Thus, we conclude that the dynamic change of copper in MSWI fly ash during heating is a commonly observed feature, and no simple relationship between the composition of fly ash and the amount of PCDD/PCDF generated or dynamic change of copper is apparent. The dynamic change of copper explained the generated amount and homologue distribution of PCDDs and PCDFs.

Histological analysis of fat body development and molting events during soldier differentiation in the damp-wood termite, Hodotermopsis sjostedti (Isoptera, Termopsidae).

The caste system of termites is well defined, with a high degree of polyphenism among colony members. Polyphenic caste characteristics are hormonally regulated, and juvenile hormone (JH) is particularly involved in caste determination, as is the case with many other social insects. In the present study, soldier differentiation in the damp-wood termite, Hodotermopsis sjostedti, was induced by treatment with a JH analog (pyriproxyfen) in order to establish the chronology of tissular modifications appearing in response to the hormone. The fat body is involved in the physiological events that prepare the insect for the molting transition. The development of the fat body started within three days after hormonal treatment, and it filled the entire abdominal cavity for about four days prior to the molt to presoldier, maintaining this state until the next molt to soldier. Fat body development was accompanied by the accumulation of protein granules in the cytoplasm, but these granules disappeared during the few days preceding the molt to presoldier. The timing of consumption of these storage proteins corresponded to the window of epidermal growth, which was conspicuous about 14 days after hormonal treatment, and synthesis of the new cuticle, which was initiated 10 days after treatment. We summarize the chronology of the histological events under hormonal control.

Kinetics on the decomposition of polychlorinated biphenyls with activated carbon-supported iron.

The process of destroying polychlorinated biphenyls (PCBs) generates exhaust gases that contain low quantities of PCBs, which cannot be disposed of easily. Activated carbon (AC) can be used to adsorb residual PCBs after disposal of high-level PCBs. We examined the chemical reactivity of AC-supported iron as a catalyst to decompose PCB-153, and varied three decomposition parameters (temperature, time and iron concentration) under an atmosphere of either air or N(2). We measured the Brunauer-Emmett-Teller (BET) surface area and pore volume of AC to assess the adsorption capacity of AC before and after decomposition. At low temperatures the adsorption process was more important than the decomposition process. The decomposition process was completed within 30 and 60 min under air and N(2), respectively. The efficiency of PCB-153 decomposition at 350 degrees C for 120 min was approximately 100.0% and 97.1% under air and N(2), respectively. Analysis of inorganic chloride ions revealed that PCB-153 was effectively destroyed during decomposition. The differences between decomposition under air and N(2) reflected differences in BET surface and pore volume.

Application of microwave-assisted extraction to the analysis of PCBs and CBzs in fly ash from municipal solid waste incinerators.

Polychlorinated biphenyls (PCBs) and chlorobenzenes (CBzs) are two classes of dioxin precursors formed in municipal solid waste incinerators (MSWIs) producing negative health effects similar to those of dioxins. Reducing the analytical time required for determining the concentrations of these compounds in MSWIs is important for quickly evaluating their importance and associated health risks. In the present study, microwave-assisted extraction (MAE) is compared with traditional Soxhlet extraction (SE) to determine the extraction efficiencies attained for PCB and CBz analysis. The efficiencies of MAE are compared with those of SE under various experimental conditions, using fly ash spiked with standards. Water is used as a safe and environmentally friendly solvent in MAE for PCB and CBz analyses and MAE has high extraction efficiency for spiked fly ash compared with that of SE. Furthermore, the extraction time and organic solvent consumption are reduced with MAE compared with SE. The optimum conditions for MAE established in this study are using a 30-ml volume of toluene/acetone (1/1) or a 15-ml volume of toluene, samples with less than 60% water content (WC), and an irradiation time of 15 min.

Screening of genes expressed in developing mandibles during soldier differentiation in the termite Hodotermopsis sjostedti.

We investigated the morphological changes accompanying soldier differentiation in the damp-wood termite Hodotermopsis sjostedti. Genes expressed in the developing mandibles, which undergo the most remarkable morphological changes during soldier differentiation, were screened using fluorescent differential display. Database searches for sequence similarities were conducted and the relative expression levels were then quantified by real-time polymerase chain reaction. Among the identified candidate genes, 12 genes were upregulated during soldier differentiation. These included genes for cuticle proteins, nucleic acid binding proteins, ribosomal proteins and actin-binding protein, which were inferred to be involved in caste-specific morphogenesis in termites.

Identification of soldier caste-specific protein in the frontal gland of nasute termite Nasutitermes takasagoensis (Isoptera: Termitidae).

The termite soldier is unique because of its defensive task in a colony. In Nasutitermitinae (family Termitidae), soldiers use in their defense frontal glands, which contain various chemical substances. To isolate the gene products related to the chemical defense, we compared the sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles of soldier heads with those of workers of the nasute termite Nasutitermes takasagoensis. We identified a 26-kDa soldier-specific protein (Ntsp1) that exists most abundantly in the dorsal head including the frontal gland. We determined the N-terminal amino acid sequence of Ntsp1, and then cloned the Ntsp1 cDNA by rapid amplification of the cDNA ends-polymerase chain reaction (RACE-PCR). A putative signal peptide was detected upstream of the N-terminus and the Ntsp1 protein showed sequence homologies with known insect secretory carrier proteins, which bind to hydrophobic ligands such as juvenile hormone, suggesting that Ntsp1 belongs to this class of proteins. Northern blot analysis confirmed that the expression level of Ntsp1 was high only in the soldier head. In addition, the localization of Ntsp1 expression was limited in epithelial cells of the frontal gland reservoir, suggesting that this protein binds to some terpenoid(s) preserved in the frontal gland reservoir.

Phylogenetic analyses of fat body endosymbionts reveal differences in invasion times of blaberid wood-feeding cockroaches (Blaberidae: Panesthiinae) into the Japanese archipelago.

Cockroaches have endosymbiotic bacteria in their fat bodies. Recent molecular phylogenetic analyses on both hosts and endosymbionts have revealed that co-evolution has occurred throughout the history of cockroaches and termites. Co-cladogenesis was also shown among closely related taxa (woodroach genus Cryptocercus; Cryptocercidae), and thus endosymbiont data are likely to be useful for biogeographical analyses. To test the possibility of co-cladogenesis among inter-and intraspecific taxa, as well as the utility of endosymbiont data for inferring biogeographical scenarios, we analyzed rRNA genes of endosymbionts of Japanese and Taiwanese Panesthiinae (Salganea and Panesthia; Blaberidae), on which phylogenetic analyses previously had been performed based on the mitochondrial genes. Statistical analyses on the topologies inferred from both endosymbiont and host mitochondria genes showed that co-cladogenesis has occurred. The endosymbiont sequences examined appear to have evolved in a clock-like manner, and their rate of evolution based on the host fossil data showed a major difference in the time of invasion of the two Japanese genera, that is congruent with the recent analyses of their mitochondrial genes.

The effect of copper speciation on the formation of chlorinated aromatics on real municipal solid waste incinerator fly ash.

A limited amount of information exists regarding the relationship between the chemical form of copper and the formation of chlorinated aromatics in fly ash. To understand the effects of the various forms of copper on the formation of chlorinated aromatics in real fly ash, we determined the chemical forms of copper present in various types of real fly ash using X-ray absorption near edge structure (XANES) and evaluated the relationship between the chemical forms of copper and the formation of chlorinated aromatics. Copper chloride hydroxide (CuCl2 x 3Cu(OH)2) and cuprous chloride (CuCl) were the predominant copper species found in real fly ash. Although pure cupric chloride (CuCl2) is known to be the most active catalyst for the formation of chlorinated aromatics under experimental conditions with synthetic fly ash, CuCl2 was not found in every real fly ash sample. The amount of copper chloride hydroxide was positively correlated with the formation of chlorinated aromatics in real fly ash and is, consequently, considered to be one of the key species involved in the formation of chlorinated aromatics.

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.

The role of termites in an equatorial rain forest ecosystem of West Malaysia : I. Population density, biomass, carbon, nitrogen and calorific content and respiration rate.

1. Density of nests, population number, biomass, nitrogen and carbon content, calorific content and respiration rate of termites were studied at Pasoh Forest, West Malaysia mainly with four dominant species of epigeous nest builders, Macrotermes carbonarius, Dicuspiditermes nemorosus type-a, type-b and Homallotermes foraminifer, to reveal their role in the ecosystem. 2. The density of nests or mounds was 15-41/ha in M. carbonarius, 60-110/ha in the two types of D. nemorosus and 85-165/ha in H. foraminifer. 3. The population number per nest or mound was about 88,000 in M. carbonarius, 45,000 in D. nemorosus type-a, 47,000 in D. nemorosus type-b and 13,000 in H. foraminifer. The population number per hectare was about 1.8x106 for M. carbonarius, 4.3x106 for D. nemorosus type-a, 5.2x106 for D. nemorosus type-b and 2.1x106 for H. foraminifer. 4. The ratio in number of workers to soldiers was 6.4 for M. carbonarius, 19.0 for D. nemorosus type-a, 23.1 for D. nemorosus type-b and 8.9 for H. foraminifer. The ratio in number of adults (workers plus soldiers) to larvae was 1.4 for M. carbonarius, 0.59 for D. nemorosus type-a, 1.76 for D. nemorosus type-b and 3.84 for H. foraminifer. 5. The number of adults per nest of D. nemorosus type-a, type-b and H. foraminifer was linearly correlated with the weight of nest on the log-log coordinates. 6. The ratio of dry body weight to live weight was 0.24-0.30 for workers, 0.19-0.29 for soldiers and 0.15-0.22 for larvae. 7. The ash content of termite body was 26-66% for workers, 2-24% for soldiers and 1-4% for larvae. 8. The carbon content ranged from 45% to 66% of ash-free dry weight and the nitrogen content from 5.6% to 12.6%. 9. The mean calorific value of termite body was 5.3 gcal/mg in workers and soldiers on an ash-free weight basis, but was greater in nymphs and winged reproductives in the nest (6.7-6.9 gcal/mg) owing probably to their large fat storage. 10. The relation of CO2 evolution rate to temperature in these termites was similar to what has been found in other insects. 11. The total biomass of the four termite species was estimated at 6.01 kg ash-free dry weight/ha, equivalent to 0.55 kg nitrogen/ha and 3.09 kg carbon/ha. 12. The role of the fungi cultivated by M. carbonarius on their fungus combs was discussed in relation to the nutrition of termites and the decomposition of leaf-litter with special reference to their nitrogen metabolism. The high nitrogen content of fungus spherules growing on fungus combs seemed to have an important bearing on the nutrition of termites. 13. It was concluded that the termites played a very important role in the organic matter decomposition cycle of Pasoh Forest.

Estimation of population productivity of Parapleurus alliaceus Germar (Orthoptera: Acridiidae) on a Miscanthus sinensis Anders. grassland : II. Population productivity in terms of dry weight.

The population production of Parapleurus alliaceus Germar on a Miscanthus sinensis Anders. grassland was studied in terms of dry weight for the purpose of measuring the role of this species in the community metabolism of the grassland. This study deals with the growth, ingestion, defaecation and assimilation of individuals. Standing crop and the amount of dead biomass were estimated using the results for population dynamics reported in Nakamura et al. (1971). Standing crops of the population at the beginning of the adult stage (August) were 17 g/100 m2 in 1968 and 6 g/100 m2 in 1969. The biomass mortality during the period from June 16 to August 23 was 1.3 g/100 m2 in 1969. The population consumed 242 g/100 m2 of M. sinensis leaves during July and August in 1968 and 97.6 g/100 m2 in 1969. The P. alliaceus population described had little effect on the primary production of M. sinensis grassland.

The role of termites in an equatorial rain forest ecosystem of West Malaysia : II. Leaf litter consumption on the forest floor.

To estimate the rate of consumption of leaf litter by termites on the forest floor of Pasoh Forest Reserve, Negeri Sembilan, West Malaysia, newly fallen leaves were marked and distributed on the ground. The loss of leaf area due to termites was determined either photometrically or visually. An average of 1.70% of the total surface area of the leaf litter disappeared per week in experiment 1 and 1.25% in experiment 2 in Plot 1, and 2.9% per day in other plots located near the mounds of Macrotermes carbonarius. The amount of leaf litter accumulation in the Ao layer was estimated at about 2.3 t/ha at Plot 1, so it was likely that an amount equivalent to about 32% of the daily leaf-litter fall was transported by M. carbonarius to their mounds in experiment 1 and 22% in experiment 2. It was considered that the termites had an important role in the detritus food chain of the ecosystem.

Estimation of population productivity of Parapleurus alliaceus Germar (Orthoptera: Acridiidae) on a Miscanthus sinensis Anders. grassland : I. Estimation of population parameters.

To assess the population productivity of the grasshopper Parapleurus alliaceus Germar in a Miscanthus sinensis Anders. grassland, estimates of population density, survival, and dilution rates were attempted using the mark-release-recapture method. No significant departure from the random-catch hypothesis, which is the basic pre-requisite for the estimation of population parameters, was discovered. A comparison between the results of two experiments, attempted simultaneously in the same area of the grassland, indicated that the accuracy of the mark-release-recapture method was acceptable for our purpose. The population trends in 1968 and 1969 showed that mortality during the early nymphal instars was remarkably low (75% and 62%, respectively, for the period from the second instar nymph to adult), and that adult longevity was greater than 10 days. The number of nymphs hatched in 1969 was smaller than 1968, and the cause of this reduction was thought to be the predation of eggs during autumn and winter.

Winged presoldiers induced by a juvenile hormone analog in Zootermopsis nevadensis: implications for plasticity and evolution of caste differentiation in termites.

To elucidate the switching mechanism of caste differentiation in termites and to examine the possible induction of soldier-reproductive intercastes experimentally, we investigated the effects of juvenile hormone on the morphologies of soldier caste by applying a juvenile hormone analog (JHA) to nymphs of the damp-wood termite Zootermopsis nevadensis (Isoptera : Termopsidae). JHA treatment for about 2 weeks induced a variety of intermediate castes, showing both alate and soldier morphological features. The principal component analysis (PCA) of those morphological characters showed that those intercastes were a deviation from the developmental line into alates to soldier differentiation, which is known to be triggered by juvenile hormone. Detailed morphological examination of the compound eyes, wing joint, and mandibles showed that those intercastes expressed soldier features, although they had started to develop alate characteristics. The morphology of the resultant intercastes seemed to be determined by the nymphal stage, at which JHA treatment was applied. The induced intercastes with exaggerated soldier-specific characteristics (e.g., mandibles) repressed alate-specific characteristics (e.g., wings), namely, the alate and soldier morphological characteristics in induced intercastes show opposite responses against the application of JHA. On the other hand, ovarian development was not suppressed by the JHA application, even in the soldier-like individuals. Naturally differentiated presoldiers also possessed developed ovarioles, although ovaries of mature soldiers were degenerated. Our results suggest that the juvenile hormone plays complicated roles in the expression of caste morphologies and ovarian development in termites.

Soldier-like intercastes in the rotten-wood termite Hodotermopsis sjostedti (Isoptera: Termopsidae).

All termite species possess a distinct sterile-soldier caste in their colonies, although reproductive soldiers, with soldier characteristics and reproductive ability, have been reported from several species of the family Termopsidae. Such intercastes have been considered the primitive-soldier caste, and based on this many researchers have discussed the evolutionary origin of termite soldiers. We investigated whether such soldier-reproductive intercastes also exist in the Japanese rotten-wood termite Hodotermopsis sjostedti. Abnormal individuals with soldier-like characteristics were found and designated as soldier-like intercastes, which appeared to have both soldier and reproductive characteristics. Based on our morphometric analyses and histological examinations, we suggest that the developmental origin of this intercaste is a pseudergate, nymph or sixth-instar larva. In addition, the intercaste was found to have relatively well-developed gonads, although mature oocytes and spermatozoa were not found in female ovaries and male seminal vesicles, respectively. We conclude that the soldier-like intercaste of H. sjostedti does not have reproductive ability, which is different from all other known soldier-like intercastes in Termopsidae.

Population structure and genetic diversity in insular populations of Nasutitermes takasagoensis (Isoptera: Termitidae) analyzed by AFLP markers.

Dispersal ability and degree of inbreeding in a population can indirectly be assessed using genetic markers. In general, it was suggested that winged termites are not able to fly distances greater than several hundred meters. Here, amplified fragment length polymorphism (AFLP) was used to analyze genetic diversity, population substructure, and gene flow among insular populations of the termite Nasutitermes takasagoensis (Isoptera: Termitidae) in the Yaeyama Islands, Okinawa, Japan. Samples were collected from 77 nests on seven islands of the Yaeyama Group. Using three primer combinations a total of 155 bands were generated with 78 (50%) polymorphic bands. Genetic distance and G(st) values among insular populations were calculated. Relatively high genetic diversity and low values of G (st), suggest there is moderate subpopulation structure. Based on these results, we discussed two possibilities; first, winged termites are able to fly over distances of several kilometers, and second, these results were obtained because insular populations share a recent common origin.