Antimicrobial Qualities of the Midgut and Haemolymph of Anasa Tristis to Different Symbiotic and Pathogenic Bacteria
499 Progress Report after 1 Semester
Fall 2013

This report follows the format of Proceedings of the Royal Society of London B
Introduction:
Many insect species harbor endosymbiotic bacteria within their tissues, organs, and organ systems that offer benefits such as nutrients, protection from pathogens or parasites, and other adaptive qualities for the host, while other bacterial strains are known to be pathogenic and to cause harm to host fitness (1, 2). In insects with a nutrient-limited diet such as leaves, wood, or plant sap, obligate symbionts aid in providing the host with additional nutrients, such as essential amino acids, that are otherwise absent in the host diet (3). Endosymbionts have also been found to have important roles in the development of insect hosts, especially during energy intensive processes such as molting (4). Recent discoveries have found that the bean bug, Riptortus pedestris, belonging to the Coreoidea superfamily, acquires symbiotic Burkholderia, not through vertical transmission from parent to offspring, but through oral acquisition from their environment (4). Also, many insect species of the Lygaeoidea and Coreoidea superfamily were found to harbor slightly different genotypes of Burkholderia within their M4 midgut, all of which were acquired from the environment in each generation (5). These discoveries are contrary to the previously understood studies of vertically transferred bacterial symbionts (4).These discoveries raise questions on the mechanisms of symbiotic establishment as well as the specificity of environmentally acquired symbionts to their insect hosts. The soil microbiota of these insects contains an enormously diverse array of bacteria and the Burkholderia account for only a small fraction of those microbes (5). The mechanism of selective acquisition of the Burholderia is largely unknown and deserves further studies.
Other studies have focused on the interactions found between pathogenic bacteria, such as Serracia marcescens, and their insect hosts. Serratia marcescens was found to not only be pathogenic to the squash bug, Anasa tristis, but also pathogenic to plants when transmitted by the bug (6). Interestingly, the plant pathogenic Serratia marcescens was not found to be pathogenic to the squash bug host transmitting it (6). It was found to be pathogenic to squash bugs once introduced in the haemolymph (7), however, the transmitted Serratia marcescens was found to be retained in other parts of the bug, possibly in the haemocoel (6). More recent studies have observed antimicrobial interactions of specific midgut regions of M4B and M4 of the midgut as well as the haemolymph of insect species such as Riptortus pedestris with symbiotic and mid-log phase Burkholderia, mid-log phase Escherichia coli, and mid-log phase Staphylococcus aureus strains (8). Aspects of the M4B and haemolymph have been observed to have antimicrobial qualities towards symbiotic bacteria as well as their prevalence in the bacterial titer (8). The M4B region has been observed to have antimicrobial activity against symbiotic Burkholderia strains suggesting roles in symbiont population control and possible utilization of symbiotic biomass for nutrients in growth (8). How Burkholderia, as opposed to other bacterial strains, are acquired into the midgut from the environment as well as how the symbiont is able to colonize the crypts of the midgut is largely unknown. In this study, we look to better understand the antimicrobial activities and bacterial control mechanisms of the haemolymph and of specific midgut sections. We investigate the antimicrobial properties of haemolymph and specific sections of the entire midgut (M1, M2, M3, M4B, M4) of the squash bug, Anasa tristis, against different symbiotic and pathogenic bacteria. These bacterial strains include two symbiotic