Parallel metatranscriptome analyses of host and symbiont gene expression in the gut of the termite Reticulitermes flavipes

<p>Abstract</p> <p>Background</p> <p>Termite lignocellulose digestion is achieved through a collaboration of host plus prokaryotic and eukaryotic symbionts. In the present work, we took a combined host and symbiont metatranscriptomic approach for investigating the digestive contributions of host and symbiont in the lower termite <it>Reticulitermes flavipes</it>. Our approach consisted of parallel high-throughput sequencing from (i) a host gut cDNA library and (ii) a hindgut symbiont cDNA library. Subsequently, we undertook functional analyses of newly identified phenoloxidases with potential importance as pretreatment enzymes in industrial lignocellulose processing.</p> <p>Results</p> <p>Over 10,000 expressed sequence tags (ESTs) were sequenced from the 2 libraries that aligned into 6,555 putative transcripts, including 171 putative lignocellulase genes. Sequence analyses provided insights in two areas. First, a non-overlapping complement of host and symbiont (prokaryotic plus protist) glycohydrolase gene families known to participate in cellulose, hemicellulose, alpha carbohydrate, and chitin degradation were identified. Of these, cellulases are contributed by host plus symbiont genomes, whereas hemicellulases are contributed exclusively by symbiont genomes. Second, a diverse complement of previously unknown genes that encode proteins with homology to lignase, antioxidant, and detoxification enzymes were identified exclusively from the host library (laccase, catalase, peroxidase, superoxide dismutase, carboxylesterase, cytochrome P450). Subsequently, functional analyses of phenoloxidase activity provided results that were strongly consistent with patterns of laccase gene expression. In particular, phenoloxidase activity and laccase gene expression are mostly restricted to symbiont-free foregut plus salivary gland tissues, and phenoloxidase activity is inducible by lignin feeding.</p> <p>Conclusion</p> <p>To our knowledge, this is the first time that a dual host-symbiont transcriptome sequencing effort has been conducted in a single termite species. This sequence database represents an important new genomic resource for use in further studies of collaborative host-symbiont termite digestion, as well as development of coevolved host and symbiont-derived biocatalysts for use in industrial biomass-to-bioethanol applications. Additionally, this study demonstrates that: (i) phenoloxidase activities are prominent in the <it>R. flavipes </it>gut and are not symbiont derived, (ii) expands the known number of host and symbiont glycosyl hydrolase families in <it>Reticulitermes</it>, and (iii) supports previous models of lignin degradation and host-symbiont collaboration in cellulose/hemicellulose digestion in the termite gut. All sequences in this paper are available publicly with the accession numbers <ext-link ext-link-id="FL634956" ext-link-type="gen">FL634956</ext-link>-<ext-link ext-link-id="FL640828" ext-link-type="gen">FL640828</ext-link> (Termite Gut library) and <ext-link ext-link-id="FL641015" ext-link-type="gen">FL641015</ext-link>-<ext-link ext-link-id="FL645753" ext-link-type="gen">FL645753</ext-link> (Symbiont library).</p

Essential oils of \u3ci\u3eCupressus funebris, Juniperus communis,\u3c/i\u3e and \u3ci\u3eJ. chinensis\u3c/i\u3e (Cupressaceae) as repellents against ticks (Acari: Ixodidae) and mosquitoes (Diptera: Culicidae) and as toxicants against mosquitoes

Juniperus communis leaf oil, J. chinensis wood oil, and Cupressus funebris wood oil (Cupressaceae) from China were analyzed by gas chromatography and gas chromatography-mass spectrometry. We identified 104 compounds, representing 66.8-95.5% of the oils. The major components were: α-pinene (27.0%), α-terpinene (14.0%), and linalool (10.9%) for J. communis; cuparene (11.3%) and δ-cadinene (7.8%) for J. chinensis; and α-cedrene (16.9%), cedrol (7.6%), and β-cedrene (5.7%) for C. funebris. The essential oils of C. funebris, J. chinensis, and J. communis were evaluated for repellency against adult yellow fever mosquitoes, Aedes aegypti (L.), host-seeking nymphs of the lone star tick, Amblyomma americanum (L.), and the blacklegged tick, Ixodes scapularis Say, and for toxicity against Ae. aegypti larvae and adults, all in laboratory bioassays. All the oils were repellent to both species of ticks. The EC95 values of C. funebris, J. communis, and J. chinensis against A. americanum were 0.426, 0.508, and 0.917 mg oil/cm2 filter paper, respectively, compared to 0.683 mg deet/cm2 filter paper. All I. scapularis nymphs were repelled by 0.103 mg oil/cm2 filter paper of C. funebris oil. At 4 h after application, 0.827 mg oil/cm2 filter paper, C. funebris and J. chinensis oils repelled ≥80% of A. americanum nymphs. The oils of C. funebris and J. chinensis did not prevent female Ae. aegypti from biting at the highest dosage tested (1.500 mg/cm2). However, the oil of J. communis had a Minimum Effective Dosage (estimate of ED99) for repellency of 0.029 ± 0.018 mg/cm2; this oil was nearly as potent as deet. The oil of J. chinensis showed a mild ability to kill Ae. aegypti larvae, at 80 and 100% at 125 and 250 ppm, respectivel

Flight performance of <i>D</i>. <i>citri</i> depending of <i>C</i>Las exposure and infection.

<p>(A) Distance covered by psyllids on the flight mill depending on their infection status and the infection status of the rearing plants, as determined by qPCR. (B) Ratio between <i>16S</i> and <i>Wg</i> genes depending on flight performance of psyllids on the flight mill. The ratio of 16S/WG increased proportionally with <i>C</i>Las DNA quantified in individual <i>D</i>. <i>citri</i>. The 16S/WG was significantly higher in infected <i>D</i>. <i>citri</i> that performed long flights (> 60s) than in infected <i>D</i>. <i>citri</i> that did not fly or performed short flights (< 60s) (<i>P</i> = 0.033, Kruskal-Wallis). A line within each boxplot indicates the median for each treatment and symbols above each boxplot indicate outliers.</p

Behavioral response of <i>D</i>. <i>citri</i> males to headspace volatiles from conspecific females exposed to <i>C</i>Las.

<p>(A) Proportion of males attracted to female odor and (B) percentage of males responding to clean air verses female odors representing each infection status. (A) Male responses are plotted against the average ratio between <i>16S</i> and <i>Wg</i> genes of the ten females of each replicate placed in the treatment arm. Females developed on <i>C</i>Las-infected citrus plants and the <i>16S</i>/<i>Wg</i> ratio indicates the amount of <i>C</i>Las DNA found per individual <i>D</i>. <i>citri</i>. Each circle indicates the proportion of males that chose the olfactometer arm with conspecific female odor for each replicate. Each replicate consisted of 20 males resulting in the test of 180 males. The dotted line and the grey area represent the average response (± SEM) of males when exposed to uninfected (control) female odors versus clean air. Control females were reared on uninfected citrus plants and were free of <i>C</i>Las. The regression equation was: <i>y</i> = 0.48 + 0.34(1 − <i>e</i>^{<i>−6</i>.<i>76x</i>}); R² = 0.87, F_2,6 = 20.31; P = 0.002. (B) Asterisks indicate significant attraction of <i>D</i>. <i>citri</i> males to female odor (**: < 0.01; ***: <0.001) compared to clean air. Different letters indicate significant differences in the proportion of male psyllids responding to the female odors among infection status treatments.</p

Dispersal behavior of <i>D</i>. <i>citri</i> depending of <i>C</i>Las exposure and infection.

<p>Dispersal of psyllids according to gender: (A) male and female; (B) female; and (C), male. Cumulative dispersal of psyllids is indicated by line graphs. Bars indicate the infection status of dispersing or non-dispersing <i>C</i>Las-exposed <i>D</i>. <i>citri</i> over four days. (A) Cumulative dispersal of <i>D</i>. <i>citri</i>. <i>C</i>Las-infected <i>D</i>. <i>citri</i> dispersed more than uninfected <i>D</i>. <i>citri</i> on day 3 (<i>P</i> = 0.011, GLM) and day 4 (<i>P</i> = 0.041, GLM), but not on days 1 and 2. (B) Cumulative dispersal of female <i>D</i>. <i>citri</i>. There were no significant differences in the dispersal and infection rates of <i>C</i>Las-exposed <i>D</i>. <i>citri</i> females. (C). Cumulative dispersal as compared between <i>C</i>Las-infected and uninfected male <i>D</i>. <i>citri</i> (<i>P</i> = 0.007, Kruskal-Wallis).The bottom graph shows the infection rates of dispersed <i>C</i>Las-exposed <i>D</i>. <i>citri</i> males. <i>C</i>Las-infected <i>D</i>. <i>citri</i> males dispersed more than uninfected <i>D</i>. <i>citri</i> males on day 3 (<i>P</i> = 0.003, GLM) and day 4 (<i>P</i> = 0.001, GLM), but not on days 1 and 2.</p

Percentage of <i>D</i>. <i>citri</i> adults tested on a flight mill that did not fly (‘non flyers’); performed only short-duration flights (‘short flyer’, < 60 s); or performed long-duration flights (‘long flyer’, > 60 s). Flight capabilities of <i>D</i>. <i>citri</i> were compared by considering <i>C</i>Las infection status of psyllids and host plants. Also included is the maximum duration of flight recorded for each category.

<p>Different letters following percentages indicate significant differences (α<0.05) among cells within the same column.</p><p>¹<i>C</i>Las⁺ and <i>C</i>Las^- refer to plants or psyllids that have tested positive (⁺) or negative (^-) for the pathogen in qPCR analyses, respectively.</p><p>²Number of psyllids tested on the flight mill for each category.</p><p>³ Average time (s) needed for psyllid to initiate a flight (non-flyers excluded).</p><p>⁴ Average velocity that <i>D</i>. <i>citri</i> flew for each category (non-flyers excluded)</p><p>Percentage of <i>D</i>. <i>citri</i> adults tested on a flight mill that did not fly (‘non flyers’); performed only short-duration flights (‘short flyer’, < 60 s); or performed long-duration flights (‘long flyer’, > 60 s). Flight capabilities of <i>D</i>. <i>citri</i> were compared by considering <i>C</i>Las infection status of psyllids and host plants. Also included is the maximum duration of flight recorded for each category.</p

Density-dependent dispersal behavior of <i>D</i>. <i>citri</i>.

<p>The dispersal of <i>D</i>. <i>citri</i> increased with increasing density. (A) Dispersal of both male and female <i>D</i>. <i>citri</i>. a = Krukal-Wallis ANOVA: p < 0.001; (B) dispersal of female <i>D</i>. <i>citri</i>; a = Krukal-Wallis ANOVA: p < 0.001; (C) dispersal of male <i>D</i>. <i>citri</i>. a = Krukal-Wallis ANOVA: p < 0.001. hd: high density (175 individuals per plant); hmd: high medium density (125 ind. p. p.); lmd: low medium density (75 ind. p. p.); ld: low density (25 ind. p. p.). Mean cumulative numbers are shown (+- SE) per day of dispersed individuals. No significant difference was found between the number of dispersed <i>D</i>. <i>citri</i> among the ld, lmd and hmd variants. However, the dispersal of <i>D</i>. <i>citri</i> in the hd variant was significantly higher than in all other variants on all dispersal days. Male and female dispersal exhibited the same pattern.</p