Host gene response to endosymbiont and pathogen in the cereal weevil Sitophilus oryzae

Background

Maternally inherited bacterial symbionts infecting arthropods have major implications on host ecology and evolution. Among them, the genus Arsenophonus is particularly characterized by a large host spectrum and a wide range of symbiotic relationships (from mutualism to parasitism), making it a good model to study the evolution of host-symbiont associations. However, few data are available on the diversity and distribution of Arsenophonus within host lineages. Here, we propose a survey on Arsenophonus diversity in whitefly species (Hemiptera), in particular the Bemisia tabaci species complex. This polyphagous insect pest is composed of genetic groups that differ in many ecological aspects. They harbor specific bacterial communities, among them several lineages of Arsenophonus, enabling a study of the evolutionary history of these bacteria at a fine host taxonomic level, in association to host geographical range and ecology.

Results

Among 152 individuals, our analysis identified 19 allelic profiles and 6 phylogenetic groups, demonstrating this bacterium's high diversity. These groups, based on Arsenophonus phylogeny, correlated with B. tabaci genetic groups with two exceptions reflecting horizontal transfers. None of three genes analyzed provided evidence of intragenic recombination, but intergenic recombination events were detected. A mutation inducing a STOP codon on one gene in a strain infecting one B. tabaci genetic group was also found. Phylogenetic analyses of the three concatenated loci revealed the existence of two clades of Arsenophonus. One, composed of strains found in other Hemiptera, could be the ancestral clade in whiteflies. The other, which regroups strains found in Hymenoptera and Diptera, may have been acquired more recently by whiteflies through lateral transfers.

Conclusions

This analysis of the genus Arsenophonus revealed a diversity within the B. tabaci species complex which resembles that reported on the larger scale of insect taxonomy. We also provide evidence for recombination events within the Arsenophonus genome and horizontal transmission of strains among insect taxa. This work provides further insight into the evolution of the Arsenophonus genome, the infection dynamics of this bacterium and its influence on its insect host's ecology.

     

Rostrum structure and development in the rice weevil Sitophilus oryzae (Coleoptera: Curculionoidea: Dryophthoridae)

A documentation and review of weevil rostrum growth is made through examination of the developmental life stages in the rice weevil Sitophilus oryzae (Linnaeus). Histological and morphological examinations are made utilizing light, fluorescent, and electron microscopy. In S. oryzae, rostral tissue begins proliferating in the late 4th instar larva and continues through to the pupal stage, with the majority of rostrum growth taking place in the prepupa. Adult cranial and rostral morphology is also reviewed, focusing on structures that may be pertinent to phylogeny reconstruction. The weevil rostrum is essentially an extension of various head sclerites that are basal to the mouthparts. Therefore, while the mouthparts are fairly similar to other Coleoptera in basic form, the head is markedly different due to its anterior extension. By understanding the more noticeable details of rostrum growth and structure, this study may serve as a foundation for comparative studies of a similar nature and as a basis for beginning research on the genetic nature of rostrum formation and evolution throughout the weevil clade.     

Influence of maternal age on the fitness of progeny in the rice weevil, Sitophilus oryzae (Coleoptera: Curculionidae)

We studied the effects of maternal age on fitness of progeny in the rice weevil, Sitophilus oryzae L. (Coleoptera: Curculionidae). Five-, 20-, and 50-d-old female rice weevils were used to study the effects of maternal age on the lifetime fecundity and longevity of their daughters. In addition, we determined the effects of maternal age on the weight and survivorship of daughters' progeny. Daughters of 5- and 20-d-old weevils lived longer, and the numbers and weights of the progeny of these daughters were higher than for daughters of 50-d-old weevils. Survivorship of immature grand-offspring of 5-, 20-, and 50-d-old female weevils was similar. None of the fitness characteristics of the daughters and grand-offspring of 5- and 20-d-old weevils that were measured differed significantly. We believe maternal age effects on rice weevil progeny fitness may at least partly be acting through maternal age effect on egg size. Individuals that developed from younger 5- and 20-d-old weevils had a greater fitness than those produced by older 50-d-old females. Our study shows maternal age is impacting life history parameters that influence population dynamics across generations. Therefore, maternal age could significantly affect population development and have far reaching implications for pest management and simulation modeling of rice weevil populations.     

Influence of olfactory stimulants on resistance/susceptibility of pearl millet, Pennisetum americanum to the rice weevil, Sitophilus oryzae

The orientation responses of the rice weevil, Sitophilus oryzae (L.) (Coleoptera: Curculionidae) as influenced by the olfactory stimulants emanating from either resistant or susceptible pearl millet, Pennisetum americanum, kernels were studied using a Y-tube olfactometer. The rice weevils were more attracted towards odours from millet grains than to blank controls and to susceptible entries more than to resistant entries, in general. However, there were exceptions. Odours from some resistant entries caused statistically the same, or even more positive orientations than those from some susceptible entries, and this suggests that the relative resistance of an entry and its olfactory attractiveness may not be strongly related, or that the resistance to infestation might be influenced by factors other than attractiveness. The partial damage of the millet kernel pericarp by abrasion slightly increased the orientational response of the weevils to odours from some susceptible and some resistant entries alike.     

Monitoring of rice weevil, Sitophilus oryzae, feeding behavior in maize seeds and the occurence of supernumerary molts in low humidity conditions

Rice weevils, Sitophilus oryzae (L.), complete their development from egg to early adulthood inside grains of wheat, rice or maize, but little is known about their feeding behavior within the seeds. An ultrasonic insect feeding monitor was used to characterize the feeding patterns of individual rice weevils as they developed in maize grains. Weevils reared in grains held at ca. 40% r.h. took longer to develop from egg to pupation than those reared in grains kept at ca. 70% r.h. Feeding patterns revealed that larvae developing in grains held at ca. 70% r.h. had only four instars, whereas supernumerary molts (five instars) occurred in certain individuals reared in grain held at ca. 40% r.h. The supernumerary molts may represent a response of the insect to stresses associated with low moisture levels in the seeds.     

Cytochrome c oxidase inhibition in the rice weevil Sitophilus oryzae (L.) by formate, the toxic metabolite of volatile alkyl formates

Volatile alkyl formates are potential replacements for the ozone-depleting fumigant, methyl bromide, as postharvest insecticides and here we have investigated their mode of insecticidal action. Firstly, a range of alkyl esters, ethanol and formic acid were tested in mortality bioassays with adults of the rice weevil, Sitophilus oryzae (L.) and the grain borer, Rhyzopertha dominica (F.) to determine whether the intact ester or one of its components was the toxic moiety. Volatile alkyl formates and formic acid caused similar levels of mortality (LC(50) 131-165 micromol l(-1)) to S. oryzae and were more potent than non-formate containing alkyl esters and ethanol (LC(50)>275 micromol l(-1)). The order of potency was the same in R. dominica. Ethyl formate was rapidly metabolised in vitro to formic acid when incubated with insect homogenates, presumably through the action of esterases. S. oryzae and R. dominica fumigated with a lethal dose of ethyl formate had eight and 17-fold higher concentrations of formic acid, respectively, in their bodies than untreated controls. When tested against isolated mitochondria from S. oryzae, alkyl esters, alcohols, acetate and propionate salts were not inhibitory towards cytochrome c oxidase (EC 1.9.3.1), but sodium cyanide and sodium formate were inhibitory with IC(50) values of 0.0015 mM and 59 mM, respectively. Volatile formate esters were more toxic than other alkyl esters, and this was found to be due, at least in part, to their hydrolysis to formic acid and its inhibition of cytochrome c oxidase.     

Distribution of the related weevil species Sitophilus oryzae and S. zeamais in Brazil

The genus Sitophilus （Coleoptera： Curculionidae） encompasses species of great economic importance as stored grain pests worldwide. Among these species, the maize and the rice weevils （Sitophilus zeamais and Sitophilus oryzae, respectively） are partic- ularly important in warmer climates. These two weevils exhibit closely morphological and ecological resemblance making difficult their proper identification and recognition of their distribution in grain-producing regions. Both species are recorded in South America and particularly in Brazil, but their respective distribution and prevalence were not yet assessed in the region. Therefore, several insect samples throughout Brazil were collected and subjected to morphological identification using male genitalia and also using molec- ular identification with species-specific primers designed for clear recognition of both the species. The primers were designed for the specific amplification of a gene fragment of the cytochrome oxidase subunit I, which exhibited high specificity during our prelimi- nary experiments with insects from six populations of known species （either S. zeamais or S. oryzae）. Both identification strategies provided the same results indicating preva- lence of the maize weevil S. zeamais throughout the country. Two hypotheses may explain such prevalence： （i） the likely host preference ofS. zeamais for maize because this is the most cultivated cereal in Brazil, and （ii） the prevalence ofS. zeamais in tropical regions as compared with S. oryzae, which is more disseminated in subtropical and temperate regions.     

Intense transpositional activity of insertion sequences in an ancient obligate endosymbiont

The streamlined genomes of ancient obligate endosymbionts generally lack transposable elements, such as insertion sequences (IS). Yet, the genome of Wolbachia, one of the most abundant bacterial endosymbionts on Earth, is littered with IS. Such a paradox raises the question as to why there are so many ISs in the genome of this ancient endosymbiont. To address this question, we investigated IS transpositional activity in the unculturable Wolbachia by tracking the evolutionary dynamics and history of ISWpi1 elements. We show that 1) ISWpi1 is widespread in Wolbachia, being present in at least 55% of the 40 sampled strains, 2) ISWpi1 copies exhibit virtually identical nucleotide sequences both within and among Wolbachia genomes and possess an intact transposase gene, 3) individual ISWpi1 copies are differentially inserted among Wolbachia genomes, and 4) ISWpi1 occurs at variable copy numbers among Wolbachia genomes. Collectively, our results provide compelling evidence for intense ISWpi1 transpositional activity and frequent ISWpi1 horizontal transmission among strains during recent Wolbachia evolution. Thus, the genomes of ancient obligate endosymbionts can carry high loads of functional and transpositionally active transposable elements. Our results also indicate that Wolbachia genomes have experienced multiple and temporally distinct ISWpi1 invasions during their evolutionary history. Such recurrent exposition to new IS invasions may explain, at least partly, the unusually high density of transposable elements found in the genomes of Wolbachia endosymbionts.     

The genome sequence of Xanthomonas oryzae pathovar oryzae KACC10331, the bacterial blight pathogen of rice

The nucleotide sequence was determined for the genome of Xanthomonas oryzae pathovar oryzae (Xoo) KACC10331, a bacterium that causes bacterial blight in rice (Oryza sativa L.). The genome is comprised of a single, 4 941 439 bp, circular chromosome that is G + C rich (63.7%). The genome includes 4637 open reading frames (ORFs) of which 3340 (72.0%) could be assigned putative function. Orthologs for 80% of the predicted Xoo genes were found in the previously reported X.axonopodis pv. citri (Xac) and X.campestris pv. campestris (Xcc) genomes, but 245 genes apparently specific to Xoo were identified. Xoo genes likely to be associated with pathogenesis include eight with similarity to Xanthomonas avirulence (avr) genes, a set of hypersensitive reaction and pathogenicity (hrp) genes, genes for exopolysaccharide production, and genes encoding extracellular plant cell wall-degrading enzymes. The presence of these genes provides insights into the interactions of this pathogen with its gramineous host.     

Mycoflora of stored rice in relation to the biological control of Sitophilus oryzae by Anisopteromalus calandrae

In this study, we determined the effect of controlling rice weevils on the mycoflora of stored brown rice by using Anisopteromalus calandrae (Howard), a parasite of the rice weevil. The mycoflora in brown rice with the parasitoid was similar to that without the parasitoid; however, the dominant mold species differed. Without the parasitoid, Aspergillus candidus Link and Penicillium islandicum Sopp were the dominant species, whereas Aspergillus penicilloides Speg. and P. islandicum were dominant when the parasitoid was present. The difference appeared to be due mainly to the difference in the grain moisture content with and without the parasitoid. A. candidus was the dominant species in the communities without the parasitoid and was not observed when the weevils were suppressed. P. islandicum dominated or co-dominated regardless of the presence of the parasitoid which suggested that P. islandicum is tolerant to low grain moisture content or is positively affected by the presence of the rice weevil.     

Purification,partial characterization,and postembryonic levels of amylases from Sitophilus oryzae and Sitophilus granarius

Amylases from adults of Sitophilus oryzae (L.) and S. granarius (L.) were purified by using a sequential procedure of ammonium sulfate precipitation, glycogen-complex formation, and ion exchange chromatography. Amylase of S. oryaze was purified 47.4-fold to a specific activity of 478 units/mg protein. One amylase unit equals 1 mg maltose hydrate produced/min at 30°C. Amylase of S. granarius was purified 85.4-fold to a specific activity of 453 units/mg protein. Amylase of S. oryzae had a Km of 0.173% for soluble starch and consisted of two anionic isozyrnes with isoelectric points of pH 3.70 and pH 3.76. Amylase of S. granarius had a Km of 0.078% for starch and was a single protein with an isoelectric point of pH 3.76. Purified amylases of both species had molecular weights of 56,000 estimated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis, were activated by chloride, and had double energies of activation calculated from Arrhenius plots. Based on fresh weights of adults feeding on whole wheat through 10 weeks of age, S. oryzae contained three-fold and eight-fold more amylase than S. granarius and S. zeamais Motschulsky, respectively. High amylase levels in S. oryzae may provide this species with an adaptive advantage when feeding on cereals containing naturally occurring amylase inhibitors.     

Specific features of vector sequences insertion in the genome of transgenic plants

Upon processing of T-DNA, both borders may be recognized incorrectly. A number of transgenic plants containing, apart from a fragment of vector DNA adjacent to the left border of T-DNA, a vector fragment inserted irrespective of it were identified. Sequence analysis confirmed that genes located beyond the boundaries of T-DNA are transferred and inserted in the genome of transgenic plants. Recombination of vector DNA was discovered among plants of the first generation after self-pollination of the original transformants.     

Specific features of vector sequences insertion in the genome of transgenic plants

Upon processing of T-DNA, both borders may be recognized incorrectly. A number of transgenic plants containing, apart from a fragment of vector DNA adjacent to the left border of T-DNA, a vector fragment inserted irrespective of it were identified. Sequence analysis confirmed that genes located beyond the boundaries of T-DNA are transferred and inserted in the genome of transgenic plants. Recombination of vector DNA was discovered among plants of the first generation after self-pollination of the original transformants.     

Lethal and sublethal effects of rice polishing process on Sitophilus oryzae (Coleoptera: Curculionidae)

The mechanical impact of rice polishingon the rice weevil, Sitophilus oryzae (L.), and the subsequent postpolishing effects associated with rice quality were studied. "Brown" rice (not polished), "White (infest then polish)" rice, and "White (polish then infest)" rice were used with different polishing process intensities and different initial weevil densities. Weevil progeny were more numerous in Brown than in White (infest then polish) or White (polish then infest) rice. Polishing corresponding to a weight loss of 11% in rice generated an immediate mortality of approximately 40%; the subsequent mortality caused by rice quality was also 40% (total >80%). A polishing process corresponding to 14 and 16% of rice weight loss generated a drastic mortality in the weevil population (>95%). The progeny per adult weevil was similar with high and low initial weevil densities. The development of weevil progeny was also delayed by about 1 wk in White (infest then polish) rice compared with Brown rice. Furthermore, weevil adults of the progeny were significantly heavier in Brown than in White (infest then polish) or White (polish then infest) rice.     

Comparing the whole-genome-shotgun and map-based sequences of the rice genome

The rice genome has now been sequenced using whole-genome-shotgun and map-based methods. The relative merits of the two methods are the subject of debate, as they were in the human genome project. In this Opinion article, we will show that the serious discrepancies between the resultant sequences are mostly found in the large transposable elements such as copia and gypsy that populate the intergenic regions of plant genomes. Differences in published gene counts and polymorphism rates are similarly resolved by considering how transposable elements affect the sequence analysis.     

Susceptibility of various developmental stages of the maize weevil, Sitophilus zeamais Motschulsky (Col., Curculionidae) to methyl iodide in brown rice

Abstract:  The efficacy of methyl iodide (MI) as a fumigant against all developmental stages of the maize weevil, Sitophilus zeamais Motsch. was investigated. Tests were conducted with concentrations of 1.5, 1.8, 2.1, 2.4, 2.7 and 3.0 mg/l, for a 6-h exposure period. Values of LC₅₀, LC₉₅ and LC₉₉ of MI for immatures and adult stages were determined. The present laboratory tests showed that MI was toxic to various life stages of S. zeamais at relatively short exposure periods. At the LC₅₀ and LC₉₅ levels, the most susceptible stage was the egg stage followed by larvae, pupae and adults (1-day mortality). The egg was found to be most susceptible to MI, requiring 0.81 and 2.16 mg/l for 50 and 99% mortality, respectively, while the adult was most tolerant, requiring 2.30 and 3.02 mg/l for 50 and 99% mortality, respectively, based on 1-day mortality count. Pupae were less susceptible to MI than egg and larvae, requiring 1.47 and 3.19 mg/l for 50 and 99% mortality, respectively. Based on the present toxicity tests, MI has the potential for use as a fumigant to control all developmental stages of the maize weevil, S. zeamais .     

Influence of Seed Size on Exploitation by the Rice Weevil, Sitophilus oryzae

Oviposition decisions and their fitness consequences for the seed parasite Sitophilus oryzae (L.) (Coleoptera: Curculionidae) were investigated. Female S. oryzae lay eggs inside seeds such as wheat [Triticum aestivum (L.)]. Because larvae develop to adult within a single seed, the resources available are determined by the behavior of the female parent and characteristics of the seed in which the egg was deposited. Females were demonstrated to lay more eggs in kernels 20 mg. Females initiated the chewing of oviposition holes in shriveled kernels but were less likely to oviposit in them. Progeny size increased with increasing seed size, but the probability of an adult emerging was not affected. Females accepted large kernels more quickly than small kernels and this contributed to increased oviposition in large kernels. The increase in the number of eggs per kernel appears to result from an increase in number of visits resulting in oviposition rather than an increase in the number of eggs laid during a visit.