Biological Role of Nardonella Endosymbiont in Its Weevil Host

Weevils constitute the most species-rich animal group with over 60,000 described species, many of which possess specialized symbiotic organs and harbor bacterial endosymbionts. Among the diverse microbial associates of weevils, Nardonella spp. represent the most ancient and widespread endosymbiont lineage, having co-speciated with the host weevils for over 125 million years. Thus far, however, no empirical work on the role of Nardonella for weevil biology has been reported. Here we investigated the biological role of the Nardonella endosymbiont for the West Indian sweet potato weevil, Euscepes postfasciatus. This insect is an experimentally tractable pest insect that can easily be reared on a natural diet of sweet potato root as well as on an agar-based artificial diet. By larval feeding on an antibiotic-containing artificial diet, Nardonella infection was effectively eliminated from the treated insects. The antibiotic-treated insects exhibited significantly lighter body weight and lower growth rate than the control insects. Then, the antibiotic-treated insects and the control insects were respectively allowed to mate and oviposit on fresh sweet potatoes without the antibiotic. The offspring of the antibiotic-treated insects, which were all Nardonella-negative, exhibited significantly lighter body weight, smaller body size, lower growth rate and paler body color in comparison with the offspring of the control insects, which were all Nardonella-positive. In conclusion, the Nardonella endosymbiont is involved in normal growth and development of the host weevil. The biological role of the endosymbiont probably underlies the long-lasting host-symbiont co-speciation in the evolutionary course of weevils

<i>Nardonella</i> infection in adult insects of <i>E. postfasciatus</i> after rifampicin treatment.

*<p>Percentage of <i>Nardonella</i> infection (number of <i>Nardonella</i>-positive insects/number of insects examined).</p>**<p>In the 1^st generation, <i>E. postfasciatus</i> was reared on artificial diet with/without the antibiotic. In the 2^nd generation, <i>E. postfasciatus</i> was reared on sweet potato without the antibiotic.</p>***<p>Two <i>Nardonella</i>-negative insects were males. Male-specific degeneration of endosymbiotic system has been known from diverse insects such as weevils, lice, mealybugs and others <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013101#pone.0013101-Buchner1" target="_blank">[4]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013101#pone.0013101-Fukatsu1" target="_blank">[54]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013101#pone.0013101-Kono1" target="_blank">[55]</a>.</p

Effects of antibiotic treatment on fitness components of <i>E. postfasciatus</i>.

<p>(A) Adult emergence, (B) body weight and (C) developmental time. The insects were reared on artificial diets with and without 0.003% rifampicin. Means, standard deviations and sample sizes are shown. Asterisks indicate statistically significant differences (* <i>P</i><0.05; ** <i>P</i><0.01).</p

Effects of <i>Nardonella</i> infection, sex, and interaction between them on fitness components of <i>E. postfasciatus</i> analyzed by generalized linear mixed models.

*<p>The two groups of insects were reared on artificial diets with and without 0.003% rifampicin, and the antibiotic-treated insects were mostly <i>Nardonella</i>-free while the untreated insects were mostly <i>Nardonella</i>-infected (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013101#pone-0013101-t002" target="_blank">table 2</a>).</p>**<p>The two groups of insects were reared on sweet potatoes without the antibiotic, and the offspring of the antibiotic-treated insects were all <i>Nardonella</i>-free while the offspring of the untreated insects all <i>Nardonella</i>-infected (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013101#pone-0013101-t002" target="_blank">table 2</a>).</p>***<p>In the 1^st generation, therefore, not exactly reflecting <i>Nardonella</i> infection but actually antibiotic treatment.</p><p>Statistically significant effects are highlighted by bold type.</p

Rearing systems for <i>E. postfasciatus</i>.

<p>(A) A mating pair of <i>E. postfasciatus</i>. (B) An artificial diet rearing dish. (C) Larvae of <i>E. postfasciatus</i> in the artificial diet. (D) A sweet potato rearing container. (E) Larvae of <i>E. postfasciatus</i> in a piece of sweet potato.</p

Effects of <i>Nardonella</i> elimination on adult body size and coloration.

<p>(A) Elytra length, (B) appearance of adult males and (C) appearance of adult females. The insects were reared on sweet potatoes without the antibiotic. In (A), means, standard deviations and sample sizes are shown, and asterisks indicate statistically significant differences (** <i>P</i><0.01).</p

Effects of <i>Nardonella</i> elimination on fitness components of <i>E. postfasciatus</i>.

<p>(A) Adult emergence, (B) body weight and (C) developmental time. The insects were reared on sweet potatoes without the antibiotic. Means, standard deviations and sample sizes are shown. Asterisks indicate statistically significant differences (** <i>P</i><0.01).</p

Effects of rifampicin doses in the artificial diet on adult emergence of <i>E. postfasciatus</i>.

*<p>Significantly different from the control (<i>P<</i>0.05; Fisher's exact probability test).</p>**<p>All were <i>Nardonella</i>-negative.</p