Tetraodon genome analysis provides further evidence for whole-genome duplication in the ray-finned fish lineage

A whole-genome duplication in the ray-finned fish lineage has been supported by the analyses of the genome sequence of the Japanese pufferfish, Fugu rubripes. Recently, genome sequence of a second teleost fish, the freshwater pufferfish, Tetraodon nigroviridis, was completed. Comparisons of long-range synteny between the Tetraodon and human genomes provided additional evidence for the whole-genome duplication in the ray-finned fish lineage. In the present study, we conducted phylogenetic analysis of the Tetraodon and human proteins to identify ray-finned fish lineage-specific (‘fish-specific’) duplicate genes in the Tetraodon genome. Our analyses provide evidence for 1087 well defined fish-specific duplicate genes in Tetraodon. We also analyzed the Fugu proteome that was predicted in the recent Fugu genome assembly, and identified 346 duplicate genes in addition to the 425 duplicates previously identified. We estimated the ages of duplicate genes using the molecular clock. The ages of duplicate genes in the two pufferfishes independently support a large-scale gene duplication around 380–400 Myr ago. In addition, a burst of recent gene duplications was evident in the Tetraodon lineage. These findings provide further evidence for a whole-genome duplication early in the evolution of ray-finned fishes, and suggest that independent gene duplications have occurred recently in the Tetraodon lineage.     

Phylogenetic analysis of the tenascin gene family: evidence of origin early in the chordate lineage

Background  

Tenascins are a family of glycoproteins found primarily in the extracellular matrix of embryos where they help to regulate cell proliferation, adhesion and migration. In order to learn more about their origins and relationships to each other, as well as to clarify the nomenclature used to describe them, the tenascin genes of the urochordate Ciona intestinalis, the pufferfish Tetraodon nigroviridis and Takifugu rubripes and the frog Xenopus tropicalis were identified and their gene organization and predicted protein products compared with the previously characterized tenascins of amniotes.     

Estimation of the Extent of Synteny Between Tetraodon nigroviridis and Homo sapiens Genomes

This paper presents a genomic comparison between 20 sequenced BACs (or fragments of BACs) from Tetraodon nigroviridis and the human genome. A total of 199 fish genes were identified by informatics resources, together with their putative human orthologues. Comparisons of the localizations in both species led to the identification of 32 syntenic regions and a minimum of 131 rearrangements in these regions that occurred during independent evolution of these species. This made it possible to estimate the rate of genomic rearrangements that occurred per million years (and per megabase). This rate is comparable to that obtained by comparison of the Fugu rubripes shotgun sequence data to human data but is significantly higher that those obtained by comparing the human genome to mammalian genomes. Overall, it suggests that genomic evolution by rearrangement is not uniform within the vertebrate group.Sequence data for the genomic BAC clones have been deposited with the DDBJ/EMBL/GenBank Data Libraries under accession numbers BX629360, BX629354, BX629355, BX629356, BX629357, BX629358, BX629359, and BX629360.     

Organization of<Emphasis Type="Italic"> Iroquois</Emphasis> genes in fish

In mammals, a total of six iroquois (Irx) genes exist, which are organized into two clusters. Here we report on the organization of all iroquois genes present in fish, using zebrafish (Danio rerio) and pufferfish (Fugu rubripes and Tetraodon nigroviridis) as examples. A total of 10 Irx genes were found in pufferfish, and 11 in zebrafish; all but one of these genes are organized into clusters (four clusters plus one isolated gene locus). The extra fish clusters result from chromosome duplication in the fish lineage, after its divergence from tetrapod vertebrates. Two of the four fish clusters are highly conserved to the ones in mammals, with regard to similarity of genes and cluster architecture. Irx genes within the other two clusters have diverged in sequence and cluster organization, suggesting functional divergence. These results will allow us to use the zebrafish system for functional and comparative studies of iroquois genes in vertebrate development.Electronic Supplementary Material Supplementary material is available in the online version of this article at Edited by D. Tautz     

DNA sequence analysis of spontaneous and γ-radiation (anoxic)-induced lacI mutations in Escherichia coliumuC122::Tn5: differential requirement for umuC at G · C vs. A · T sites and for the production of transversions vs. transitions

Escherihica coliumC122::Tn5 cells were γ-radiated (¹³⁷Cs, 750 Gy, under N₂), and lac-constitutive mutants were produced at 36% of the wild-type level (the umC strain was not deficient in spontaneous mutagenesis, and the mutational spectrum determined by sequencing 263 spontaneous lacI^d mutations was very similar to that for the wild-type strain). The specific nature of the umC strain's partial radiation was determined by sequencing 325 radiation-induced lacI^d mutations. The yields of radiation-induced mutation classes in the umC strain (as a percentage of the wild-type yield) were: 80% for A · T → G · C transitions, 70% for multi-base additions, 60% for single-base deletions, 53% for A · T → C · G transversions, 36% for G · C → A · T transitions, 25% for multi-base deletions, 21% for A · T → T · A transversions, 11% for G · C → C · G transversions, 9% for G · C → T · A transversions and 0% for multiple mutations. Based on these deficiencies and other factors, it is concluded that the umC strain is near-normal for A · T → G · C transitions, single-base deletions and possibly A · T → C · G transversions; is generally deficient for mutagenesis at G · C sites fro transversions, and is grossly deficient in multiple mutations. Damage at G · C sites seems more difficult for translesion DNA synthesis to bypass than damage at A · T sites, and especially when trying to produced a transversion. The yield of G · C → A · T transitions in the umC strain *36% of the wild-type level) argues that a basic sites are involved in no more than 64% of γ-radiation-induced base substitutions in the wild-type strain. Altogether, these data suggest that the UmuC and UmuD′ proteins facilitate, rather than being absolutely required for, translesion DNA synthesis; with the degree of facilitation being dependent both on the nature of the noncoding DNA damage, i.e., at G · C vs A · T sites, and on the nature of the misincorporated base, i.e., whether it induces transversions or transitions.     

<Emphasis Type="Italic">Takifugu obscurus</Emphasis> is a euryhaline fugu species very close to <Emphasis Type="Italic">Takifugu rubripes</Emphasis> and suitable for studying osmoregulation

Background  

The genome sequence of the pufferfish Takifugu rubripes is an enormously useful tool in the molecular physiology of fish. Euryhaline fish that can survive both in freshwater (FW) and seawater (SW) are also very useful for studying fish physiology, especially osmoregulation. Recently we learned that there is a pufferfish, Takifugu obscurus, common name "mefugu" that migrates into FW to spawn. If T. obscurus is indeed a euryhaline fish and shares a high sequence homology with T. rubripes, it will become a superior animal model for studying the mechanism of osmoregulation. We have therefore determined its euryhalinity and phylogenetic relationship to the members of the Takifugu family.     

Freshwater fugu or pufferfishes of the genusTetraodon from the Mekong basin, with descriptions of two new species

The fresh waters of the Mekong basin are inhabited by five species of the pufferfish genusTetraodon Linnaeus, 1758 (sensu lato): the widely distributedT. leiurus Bleeker, 1851; two previously described species endemic to the Mekong,T. baileyi Sontirat, 1989 andT. suvattii Sontirat & Soonthornsathit, 1985; one new species endemic to the Mekong basins.Tetraodon baileyi has now been collected numerous times but only in or near rapids;T. abei, T. barbatus andT. suvattii are often but not exclusively associated with rapids or at least swift flowing streams; whileT. leiurus is found in almost all habitats except rapids.Tetraodon abei andT. barbatus are closely related toT. leiurus, from which they differ most strikingly in coloration.Tetraodon survattii differs from all of the others in having a strongly upturned mouth and light and dark stripes or spots radiating outwards from the eye.Tetraodon baileyi, the most distinctive Mekong pufferfish, has head and body more or less extensively covered with dermal cirri and is entirely scaleless.     

Dominant fungi in the rhizosphere of established tea bushes and their interaction with the dominant bacteria under in situ conditions

Species of Penicillium and Trichoderma were found to dominate the rhizosphere of established tea bushes in a detailed study conducted from various tea growing locations in India. Penicillium erythromellis, P. janthinellum, P. raistrickii, Trichoderma pseudokoningii and T. koningii were found to be closely associated with tea roots. While seasonal fluctuation was observed in the case of Penicillium spp., the population of Trichoderma spp. showed less variation during the year. Both species were sensitive to low temperatures. In general, fungi associated with the tea rhizosphere were found to prefer a mesophillic temperature range (15 °C to 35 °C). The dominant species of Penicillium and Trichoderma also exhibited tolerance to lower temperatures, i.e., 5 to 10 °C on agar plates. Most fungi were able to grow in a wide range of pH (4 to 12). Lowering of soil pH in the rhizosphere of tea bushes was positively correlated with the age of the bush and may have affected the development of a specific microbial community in the rhizosphere.The populations of Penicillium and Trichoderma species were inversely correlated with the populations of two most dominant rhizosphere bacteria, Bacillus subtilis and B. mycoides. Both Bacillus species have been shown to have antagonistic activity against these two fungi under in vitro conditions. The present study demonstrates the existence of a similar antagonism under in situ conditions in the rhizosphere of established tea bushes.     

Conidial germination and germ tube elongation of Phomopsis amaranthicola and Microsphaeropsis amaranthi on leaf surfaces of seven Amaranthus species: Implications for biological control

Microsphaeropsis amaranthi and Phomopsis amaranthicola are potential biological control agents for several Amaranthus species. In an effort to understand the initial infection processes with these pathogens, a study was conducted of the conidial germination and germ tube length (μm) on the weed leaf surfaces at 21 °C and 28 °C. Weeds included Amaranthus rudis, A. palmeri, A. powellii, A. retroflexus, A. spinosus, A. hybridus, and A. albus. For P. amaranthicola, conidial germination and germ tube length varied among the seven weed species at both temperatures, while for M. amaranthi the differences in germ tube lengths were significant among weed species only at 21 °C. While the conidia of M. amaranthi and P. amaranthicola germinated on the leaf surfaces of all seven weed species, temperature appeared to impact the number and length of germ tubes on the leaf surfaces. The percentage of germinated conidia and the length of germ tubes at both temperatures were often greater for M. amaranthi than for P. amaranthicola. In order for the fungal pathogen to successfully infect and kill a weedy host, conidia must germinate and form a germ tube, two processes that vary with host species and temperature for M. amaranthi and P. amaranthicola. The extent to which successive infection processes, e.g., penetration, invasion and colonization, contribute to host specificity warrants study.     

Toxicity of pufferfish Takifugu rubripes cultured in netcages at sea or aquaria on land

Marine pufferfish (family Tetraodontidae) are believed to accumulate tetrodotoxin (TTX) mainly in liver and ovary through the food chain by ingesting TTX-bearing organisms such as starfish, gastropods, crustacean, flatworms, ribbonworms, etc. Consequently, it is hypothesized that non-toxic pufferfish can be produced if they are cultured with TTX-free diets in netcages at sea or aquaria on land, where the invasion of TTX-bearing organisms is completely shut off. To confirm this hypothesis, more than 5000 specimens of the pufferfish (“torafugu”, Takifugu rubripes) cultured in such manners for 1–3 years were collected from several locations in Japan during 2001–2004, and toxicity of their livers and some other parts was examined according to the Japanese official mouse assay method for TTX. In addition, typical specimens were submitted to LC/MS analysis. The results showed that all the livers and other parts tested were ‘non-toxic’ in both of the mouse assay (less than 2 MU/g) and LC/MS analysis (less than 0.1 MU/g). Thus, it is undoubtedly confirmed that pufferfish are intoxicated through the food chain, and non-toxic pufferfish can be successfully produced by netcage or land culture. The livers from these fish can be used with safety as a Japanese traditional food “fugu-kimo” (puffer liver).     

Molecular cloning and expression of retinoic-acid synthesizing enzyme raldh2 from Takifugu rubripes

Retinaldehyde dehydrogenases (raldhs) synthesize retinoic acid (RA), which is required for pattern formation and organogenesis during embryogenesis. To elucidate the common role of RA on vertebrate embryos, we first sought to clone a homologous gene to human raldh2 from fugu, Takifugu rubripes. We cloned a 1837 bp cDNA that encodes fugu raldh. The deduced amino acid sequence of the fugu raldh comprises 502 amino acids. The fugu Raldh showed highest sequence identity to zebrafish, Danio rerio, Raldh2 (79.9%). The fugu Raldh also showed high sequence identity to other vertebrate Raldh2: Xenopus laevis (77.2%), human (77.4%), mouse (74.3%) and chick (73.9%). Comparative genomic analysis showed that the gene arrangement around fugu raldh agreed with that of human raldh2. Fugu raldh mRNA was expressed through embryogenesis similarly to raldh2 in other vertebrates. These results and phylogenetic analyses suggest that pufferfish raldh is a fugu orthologue of other species' raldh2.     

Structural comparison of promoter and coding sequence of type I collagen alpha 1 chain gene duplicates between zebrafish and flounder/fugu lineages

Alpha 1 chain (Colα1(I)) and alpha 2 chain (Colα2(I)) are universal components of type I collagen in tetrapods, but rainbow trout (Oncorhynchus mykiss) and zebrafish (Danio rerio) have a third: alpha 3 chain (Colα3(I)). This study tests whether Colα3(I) is a duplicate of Colα1(I) by whole-genome duplication (WGD) that occurred early in the ray-fin fish lineage. We also examine how their promoter sequence was modified after WGD. We cloned Colα1(I), Colα2(I) and Colα3(I) cDNAs and their promoters from flounder (Paralichthys olivaceus) and obtained corresponding sequences from the genome databanks of two pufferfishes Takifugu rubripes and Tetraodon nigroviridis, by BLAST-Search using flounder sequences. Phylogenetic analysis of N-terminal sequences of ca. 100 amino acids, including signal peptide and N-propeptide sequences before short triple helical domain, indicates that Colα3(I), found only in teleosts, is a duplicate of Colα1(1) by WGD. Colα1(I) and Colα3(I) genes begin to be transcribed at different stages of Takifugu embryogenesis, suggesting that their structure of promoter is modified differently after WGD. In flounder, Takifugu and Tetraodon, the structure of proximal region of promoter is highly conserved within Colα1(I) and within Colα3(I); no homology is apparent except for the TATA element motif between Colα1(I) and Colα3(I) of each species. Unexpectedly, zebrafish Colα1(I) promoter is more homologous to Colα3(I) of flounder and fugu than Colα1(I) is. These results suggest that each duplicated Colα1(I) gene promoter inherited a unique structure after WGD, but the manner of modification differed between the phylogenetically separated zebrafish and flounder/pufferfish lineages.     

Characterization of biocontrol traits in the entomopathogenic nematode Heterorhabditis georgiana (Kesha strain), and phylogenetic analysis of the nematode’s symbiotic bacteria

Our objective was to estimate the biocontrol potential of the recently discovered entomopathogenic nematode species Heterorhabditis georgiana (Kesha strain). Additionally, we conducted a phylogenetic characterization of the nematode’s symbiotic bacterium. In laboratory experiments, we compared H. georgiana to other entomopathogenic nematodes for virulence, environmental tolerance (to heat, desiccation, and cold), and host seeking ability. Virulence assays targeted Acheta domesticus, Agrotis ipsilon, Diaprepes abbreviatus, Musca domestica, Plodia interpunctella, Solenopsis invicta, and Tenebrio molitor. Each assay included H. georgiana and five or six of the following species: Heterorhabditis floridensis, Heterorhabditis indica, Heterorhabditis mexicana, Steinernema carpocapsae, Steinernema feltiae, Steinernema rarum, and Steinernema riobrave. Environmental tolerance assays included Heterorhabditis bacteriophora, H. georgiana, H. indica, S. carpocapsae, S. feltiae, and S. riobrave (except cold tolerance did not include S. carpocapsae or S. riobrave). Host seeking ability was assessed in H. bacteriophora, H. georgiana, S. carpocapsae, and Steinernema glaseri, all of which showed positive orientation to the host with S. glaseri having greater movement toward the host than S. carpocapsae (and the heterorhabditids being intermediate). Temperature range data (tested at 10, 13, 17, 25, 30 and 35 °C) indicated that H. georgiana can infect Galleria mellonella between 13 and 35 °C (with higher infection at 17–30 °C), and could reproduce between 17 and 30 °C (with higher nematode yields at 25 °C). Compared with other nematode species, H. georgiana expressed low or intermediate capabilities in all virulence and environmental tolerance assays indicating a relatively low biocontrol potential. Some novel observations resulted from comparisons among other species tested. In virulence assays, H. indica caused the highest mortality in P. interpunctella followed by S. riobrave; S. carpocapsae caused the highest mortality in A. domesticus followed by H. indica; and S. riobrave was the most virulent nematode to S. invicta. In cold tolerance, S. feltiae exhibited superior ability to cause mortality in G. mellonella (100%) at 10 °C, yet H. bacteriophora and H. georgiana exhibited the ability to produce attenuated infections at 10 °C, i.e., the infections resumed and produced mortality at 25 °C. In contrast, H. indica did not show an ability to cause attenuated infections. Based on the phylogenetic analysis, the bacterium associated with H. georgiana was identified as Photorhabdus luminescens akhurstii.     

Biological control of Tipula paludosa (Diptera: Nematocera) using entomopathogenic nematodes (Steinernema spp.) and Bacillus thuringiensis subsp. israelensis

Tipula paludosa (Diptera: Nematocera) is the major insect pest in grassland in Northwest Europe and has been accidentally introduced to North America. Oviposition occurs during late August and first instars hatch from September until mid-October. Laboratory and field trials were conducted to assess the control potential of entomopathogenic nematodes (EPN) (Steinernema carpocapsae and S. feltiae) and Bacillus thuringiensis subsp. israelensis (Bti) against T. paludosa and to investigate whether synergistic effects can be exploited by simultaneous application of nematodes and Bti. Results indicate that the early instars of the insect are most susceptible to nematodes and Bti. In the field the neonates prevail when temperatures tend to drop below 10 °C. S. carpocapsae, reaching >80% control, is more effective against young stages of T. paludosa than S. feltiae (<50%), but the potential of S. carpocapsae might be limited by temperatures below 12 °C. Mortality of T. paludosa caused by Bti was not affected by temperature even at 4 °C but the lethal time increased with decreasing temperatures. Synergistic effects of Bti and EPN against T. paludosa were observed in 3 out of 10 combinations in laboratory assays but not in a field trial. The potential of S. carpocapsae was demonstrated in field trials against early instars in October reaching an efficacy of >80% with 0.5 million nematodes m⁻² at soil temperatures ranging between 3 and 18 °C. Results with Bti were strongly influenced by the larval stage and concentration. Against early instars in autumn between 74 and 83% control was achieved with 13 kg ha⁻¹ Bti of 5,700 International Toxic Units (ITUs) and 20 kg ha⁻¹ of 3,000 ITUs. Applications in spring against third and fourth instars achieved between 0 and 32% reduction. The results indicate that application of Bti and nematodes will only be successful and economically feasible during the early instars and that the success of S. carpocapsae is dependent on temperatures >12 °C. Synergistic effects between S. carpocapsae and Bti require more detailed investigations in the field to determine maximal effect.     

Selection of Beauveria bassiana isolates for control of the whiteflies Bemisia tabaci and Trialeurodes vaporariorum on the basis of their virulence, thermal requirements, and toxicogenic activity

As part of a 3-fold approach to select potential mycoinsecticides for whitefly control, we evaluated infectivity, thermal requirements, and toxicogenic activity of the entomopathogenic fungus Beauveria bassiana (Ascomycota: Clavicipitaceae) under laboratory conditions. Twenty-five native B. bassiana isolates and a commercially available mycoinsecticide (based on B. bassiana) were evaluated for virulence to fourth instar nymphs of sweetpotato whitefly, Bemisia tabaci, and greenhouse whitefly, Trialeurodes vaporariorum, at a concentration of 1 × 10⁷ conidia/ml. All isolates were pathogenic for both whitefly species, whereas mortality rates varied from 3 to 85%. A second series of bioassays was conducted on 10 selected isolates using four 10-fold concentrations ranging from 1 × 10⁵ to 1 × 10⁸ conidia/ml. Median lethal concentrations (LC₅₀) of the four most virulent isolates varied from 1.1 × 10⁵ to 6.2 × 10⁶ conidia/ml and average survival time (AST) of treated nymphs from 5.9 to 7.4 days. T. vaporariorum were significantly more susceptible to all B. bassiana isolates than B. tabaci. The thermal biology of the eight most virulent isolates to both whitefly species was investigated at six temperatures (10–35 °C). The colony radial growth rate was estimated from the slope of the linear regression of colony radius on time and data were then fitted to a modified generalized β function that accounted for 90.5–99.3% of the data variance. Optimum temperatures for extension rate ranged from 23.1 to 27.1 °C, whereas maximum temperatures for fungal growth varied from 31.8 to 36.6 °C. On the basis of their virulence and thermal requirements, three isolates showed promise as candidates for whitefly management in Mediterranean greenhouses. Whilst in vitro production of macromolecular compounds toxic to Galleria mellonella larvae was not a requisite for virulence, ASTs of larvae injected with Sephadex G-25 fractions from candidate isolates ranged from 1.4 to 3.7 days compared with 5–6 days for non-toxic G-25 fractions. In addition, proteinase K treatment significantly reduced their toxic activity suggesting that they were proteins and revealing the potential of these isolates to be further improved through biotechnology to kill the pest more quickly.     

Thermal requirements for the embryonic development of Periplaneta americana (L.) (Dictyoptera: Blattidae) with potential application in mass-rearing of egg parasitoids

The embryonic development of oothecae of Periplaneta americana was evaluated under four different constant temperatures (5, 10, 15, 20, 25, 30, and 35 °C) and also at different exposure times at <5 °C. Their suitability as hosts after the treatment for the parasitoids Evania appendigaster and Aprostocetus hagenowii was also assessed. Temperatures of 5, 10, 15, and 35 °C adversely affected the development of the cockroaches, and exposure times to <5 °C longer than 5 days sufficed to kill all the embryos in the oothecae. The lower thermal threshold for complete development of P. americana was estimated to be 6.8 °C, with a required total amount of 900.9 degree-days. Cold-killed oothecae were still fit for the development of parasitoids. Parasitism rates of A. hagenowii were higher than those of E. appendigaster, although with lower emergence rates. Our results can be useful in aiding mass-rearing of these parasitoids for biological control programmes of P. americana, and may help forecast the time of emergence of nymphs of American cockroaches in infested areas.     

Entomopathogenic fungi of Eurygaster integriceps Puton (Hemiptera: Scutelleridae): collection and characterization for development

Exploratory activities were done in Syria, Turkey, Iran, Uzbekistan, Kazakhstan, The Kyrghyz Republic, and Russia to locate entomopathogenic fungi of Eurygaster integriceps. Isolates from the entomopathogenic genera Beauveria, Paecilomyces, and Verticillium were collected. Beauveria bassiana was the most commonly recovered species. Thirty-one isolates of the 221 recovered were examined at 20, 25, 30, and 35 °C for 20 days for growth and sporulation. Growth and sporulation were generally highest at 25 °C. None of the isolates grew at 35 °C, and at 30 °C growth was retarded with no conidia being produced. Single- and multiple-concentration bioassays were conducted on greenhouse-grown wheat plants and in pine litter to evaluate virulence of fungi from several sources to E. integriceps. When tested at a single concentration, mortality after 15 days ranged from 66 to >95% in the litter assays and 50 to 91% in the plant assays. There was a distinct concentration response for most of the isolates tested in the multiple-concentration assay, particularly in the in-litter environment. In litter, mortality tended to develop earlier than in on-plant assays. Several isolates of B. bassiana and one Metarhizium anisopliae displayed consistently high virulence against E. integriceps and were more virulent than two commercial strains. Our results demonstrate the potential of entomopathogenic fungi for management of E. integriceps in overwintering sites and in wheat fields.     

Simultaneous determination of pyridostigmine bromide, N,N-diethyl-m-toluamide, permethrin, and their metabolites in rat plasma and urine by high-performance liquid chromatography

A rapid and simple method was developed for the separation and quantification of the anti nerve agent drug pyridostignmine bromide (PB; 3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide) its metabolite N-methyl-3-hydroxypyridinium bromide, the insect repellent DEET (N,N-diethyl-m-toluamide), its metabolites m-toluamide and m-toluic acid, the insecticide permethrin (3-(2,2-dichloro-ethenyl)-2,2-dimethylcyclopropanecarboxylic acid(3-phenoxyphenyl)methylester), and two of its metabolites m-phenoxybenzyl alcohol, and m-phenoxybenzoic acid in rat plasma and urine. The method is based on using C₁₈ Sep-Pak^® cartridges for solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) with reversed-phase C₁₈ column, and gradient UV detection ranging between 208 and 230 nm. The compounds were separated using gradient of 1 to 99% acetonitrile in water (pH 3.20) at a flow-rate ranging between 0.5 and 1.7 ml/min in a period of 17 min. The retention times ranged from 5.7 to 14.5 min. The limits of detection were ranged between 20 and 100 ng/ml, while limits of quantitation were 150–200 ng/ml. Average percentage recovery of five spiked plasma samples were 51.4±10.6, 71.1±11.0, 82.3±6.7, 60.4±11.8, 63.6±10.1, 69.3±8.5, 68.3±12.0, 82.6±8.1, and from urine 55.9±9.8, 60.3±7.4, 77.9±9.1, 61.7±13.5, 68.6±8.9, 62.0±9.5, 72.9±9.1, and 72.1±8.0, for pyridostigmine bromide, DEET, permethrin, N-methyl-3-hydroxypyridinium bromide, m-toluamide, m-toluic acid, m-phenoxybenzyl alcohol and m-phenoxybenzoic acid, respectively. The relationship between peak areas and concentration was linear over the range between 100 and 5000 ng/ml. This method was applied to analyze the above chemicals and metabolites following their administration in rats.