Engineering a Culturable Serratia symbiotica Strain for Aphid Paratransgenesis

Abstract

Aphids are global agricultural pests and important models for bacterial symbiosis. To date, none of the native symbionts of aphids have been genetically manipulated, which limits our understanding of how they interact with their hosts. Serratia symbiotica CWBI-2.3T is a culturable, gut-associated bacterium isolated from the black bean aphid. Closely related Serratia symbiotica strains are facultative aphid endosymbionts that are vertically transmitted from mother to offspring during embryogenesis. We demonstrate that CWBI-2.3T can be genetically engineered using a variety of techniques, plasmids, and gene expression parts. Then, we use fluorescent protein expression to track the dynamics with which CWBI-2.3T colonizes the guts of multiple aphid species, and we measure how this bacterium affects aphid fitness. Finally, we show that we can induce heterologous gene expression from engineered CWBI-2.3T in living aphids. These results inform the development of CWBI-2.3T for aphid paratransgenesis, which could be used to study aphid biology and enable future agricultural technologies. IMPORTANCE Insects have remarkably diverse and integral roles in global ecosystems. Many harbor symbiotic bacteria, but very few of these bacteria have been genetically engineered. Aphids are major agricultural pests and an important model system for the study of symbiosis. This work describes methods for engineering a culturable aphid symbiont, Serratia symbiotica CWBI-2.3T. These approaches and genetic tools could be used in the future to implement new paradigms for the biological study and control of aphids.

Vertical Transmission at the Pathogen-Symbiont Interface: Serratia symbiotica and Aphids

“…Based on previous studies, S. symbiotica CWBI-2.3 T acts as a gut pathogen in its original host, the black bean aphid (44). In both black bean aphid and pea aphid hosts, S. symbiotica CWBI-2.3 T appears to be restricted to the gut and is not observed infecting hemolymph (44,47,48). To determine whether S. symbiotica CWBI-2.3 T and HB1 can persist and act as pathogens in the hemolymph of pea aphids, we injected fourth-instar pea aphids with tagged strain CWBI-GFP or HB1-GFP at two doses: low (;80 cells injected, 3 replicate trials per treatment) and high (;800 cells injected, 1 trial per treatment).…”

“…Second, these strains do not appear to undergo vertical transmission in natural infections. Following ingestion by black bean or pea aphids, S. symbiotica CWBI-2.3 T is not subsequently detected in hemolymph or embryos but is present in the gut and in honeydew, suggesting that the dominant route of transmission for this strain is fecal-oral (33,43,47,48). We injected CWBI-2.3 T and HB1 into hemolymph to determine if they are nonetheless capable of transovarial transmission in pea aphids.…”

A Genetically Modified Anti-Plasmodium Bacterium Is Harmless to the Stingless Bee Partamona Helleri

“…In general, the genus Serratia is non-pathogenic and is constitutively found in the guts of many arthropods, including mosquitoes, bees, sand ies, ticks, beetles, and aphids [4][5][6][7][8][9]45]. In our experiments, ingestion of the two tested bacterial strains was not harmful to the bees.…”

“…For instance, strain Ss1 is pathogenic when in high abundance in honeybees [33,37], whereas RPWL1 can be pathogenic to the beetle Rhynchophorus ferrugineus [46]. Conversely, a genetically modi ed Serratia symbiotica did not affect the tness or survival of aphids, suggesting that it is an important future paratransgenic tool for the control of agricultural pests [9].…”

“…The facultative aerobic and gram-negative rod-shaped bacteria of the genus Serratia (Enterobacteriaceae) are common components of the midgut microbiota. This genus is a symbiont of many arthropods, such as mosquitoes, bees, sand ies, ticks, and aphids [4][5][6][7][8][9].…”