Multilocus sequence typing of the invasive pest Halyomorpha halys (Hemiptera: Pentatomidae) and associated endosymbiont reveals unexplored diversity

: Halyomorpha halys is an invasive pest affecting a wide range of crops in many regions of the world. Rapid and cost-effective methods to reconstruct its invasion routes are crucial for implementing strategies to prevent further spread. The mitochondrial markers COI and COII and the pseudogene ΔybgF of the primary symbiont "Candidatus Pantoea carbekii" have been analyzed to track the spread of H. halys. However, these markers do not provide sufficient resolution to fully elucidate invasion routes. Here, H. halys individuals from native and invasive populations were analyzed to identify new DNA markers and evaluate their effectiveness in a multilocus sequence typing (MLST) framework. Three new nuclear markers for H. halys (Hh_KsPi, Hh_UP1, Hh_D3PDh) and three new markers for P. carbekii (Pc_TamA, Pc_SucA, Pc_SurA) were identified. Hh_D3PDh was the most informative marker for H. halys, describing two more haplotypes than COI. By integrating Hh_D3PDh with mitochondrial markers, 30 distinct haplotypes were identified, with each of the populations studied exhibiting multiple haplotypes. Pc_SucA was the most informative symbiont marker, and when all P. carbekii markers were combined, symbiont diversity was greatly increased. The low network specialization between the novel nuclear markers and both mitochondrial and symbiont markers underlined the higher power of nuclear markers. Interestingly, perfect network specialization between H. halys COI and symbiont markers was found in populations from invaded areas, suggesting that some holobiont variants may contribute to enhanced invasive ability. A MLST workflow is proposed as a new tool for population genetics analysis and reconstruction of H. halys invasion.