Molecular Markers for weevil (Sitophilus zeamais) population genetics




Huansheng Cao

Introduction

Weevil species of the genus of Sitophilus (Coleoptera: Curculionidae), are pests of stored cereals around the world. Maize weevils (S. zeamais) and rice weevils (S. oryzae), are two of the three major pest species and much effort has been made to control these pests. However the population genetics of Sitophilus weevils remains unexplored. Sitophilus weevils are also hosts to many endosymbiotic bacteria. Endosymbiosis is a ubiquitous interspecific association and plays significant roles in many ecological and evolutionary processes. Weevils provide symbionts with a stable environment and some metabolites, while the symbiont provides the weevil with nutrients deficient in their diet. Compared with the extensive studies on their endosymbionts, our knowledge of the weevil is quite limited. Populations in different regions would probably feed on different diets characteristic of their habitat environment. As a consequence, the endosymbionts residing within them may also differ in their ability to synthesize required nutrients. An endosymbiont undergoes substantial genomic change following host-restriction, and therefore the association could be distinct among isolated weevil populations. Some weevil populations have shown genetic structuring across large spatial scales. Thus, a phylogenetic analysis of weevil populations would not only aid pest management, but it would also further our understanding of the weevil-bacterium endosym- biosis.

In this pilot study, we tested the COI (cytochrome oxidase subunit one) and COI-TL2 (sequence spanning COI and leucine tRNA gene 2) regions for their utility in examining the population genetics of S. zeamais. A rice weevil (S. oryzae) was included in the study as an outgroup for the construction of a phylogenetic tree.

Figures


Figure 1-Locations of weevil (S. zeamais) populations used in this study.


Figure 2-Partial COI and COI-CTL2 gene fragments amplified from four individual weevils (S. zeamais) harvested in four states and a S. oryzae individual. S. zeamais partial COI and COI-CTL2 segments: IN (lanes 1 and 6), KS (2 and 7), NE ( 3 and 8), PA (5 and 10); S. oryzae partial COI and COI-CTL2 segments (lanes 4 and 9); negative control: lanes 12 (COI) and 13 (COI-CTL2); ladder (lane 11).


Figure 3-Alignment of partial COI (A) and COI-TL2 (B) sequences amplified from four individual weevils (S. zeamais) harvested in different states and a S. oryzae individual. * indicates identity among all five sequences. The base change in NE S. Zeamais sequence is pointed by arrow. Base changes in S. oryzae COI and COI-TL2 sequence are boxed.


Figure 4-Phylogenetic relationship between S. zeamais populations and between S. zeamais and S. oryzae based on the sequence of the integrated 1304-bp fragment. This tree was constructed using minimum evolution calculation with MEGA 3.1.


Summary

Sitophilus weevils are cosmopolitan pests of stored grain that harbor bacterial endosymbionts. Therefore an examination of weevil population genetics and phylogeny would provide insight into both pest management and symbiotic evolution. We tested molecular markers in mitochondrial DNA for their utility in such studies. Two overlapping gene fragments, corresponding to partial cytochrome subunit one (COI) gene and partial COI and leucine tRNA 2 genes (COI-TL2), were amplified by PCR using two degenerate primer sets from individual weevils (S. zeamais) collected in Indiana (IN), Kansas (KS), Nebraska (NE), and Pennsylvania (PA), and an S. oryzae individual. A 644-bp partial COI fragment and an 831-bp partial COI-TL2 were purified and sequenced. The two overlapping fragments were pieced into one long consecutive sequence. Populations from IN, KS, and PA showed sequence homology while NE population differed at a single base. Sequence differed substantially between S. zeamais and S. oryzae, with a 14% sequence divergence. Phylogenetic tree analysis reflected intra- and interspecific relationships. These markers would be useful in S. zeamais population genetics and interspecific phylogenetic studies.

For more details, please refer to the attached full paper.

Full Paper

Acknowledgments

I would like to thank Dr. Berish Rubin for his instructions and permission to use his lab, Joe Frezzo and Leleesha Samaraweera for their time and assistance in the lab. Thanks also go to Dr. Gordon Plague and Kevin Dougherty for discussion of the experiment and providing the weevils.


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