Honey bee functional genomics using symbiont-mediated RNAi.

Honey bees are indispensable pollinators and model organisms for studying social behavior, development and cognition. However, their eusociality makes it difficult to use standard forward genetic approaches to study gene function. Most functional genomics studies in bees currently utilize double-stranded RNA (dsRNA) injection or feeding to induce RNAi-mediated knockdown of a gene of interest. However, dsRNA injection is laborious and harmful, and dsRNA feeding is difficult to scale cheaply. Further, both methods require repeated dsRNA administration to ensure a continued RNAi response. To fill this gap, we engineered the bee gut bacterium Snodgrassella alvi to induce a sustained host RNA interference response that reduces expression of a targeted gene. To employ this functional genomics using engineered symbionts (FUGUES) procedure, a dsRNA expression plasmid is cloned in Escherichia coli using Golden Gate assembly and then transferred to S. alvi. Adult worker bees are then colonized with engineered S. alvi. Finally, gene knockdown is verified through qRT-PCR, and bee phenotypes of interest can be further assessed. Expression of targeted genes is reduced by as much as 50-75% throughout the entire bee body by 5 d after colonization. This protocol can be accomplished in 4 weeks by bee researchers with microbiology and molecular cloning skills. FUGUES currently offers a streamlined and scalable approach for studying the biology of honey bees. Engineering other microbial symbionts to influence their hosts in ways that are similar to those described in this protocol may prove useful for studying additional insect and animal species in the future.

Symbionts shape host innate immunity in honeybees.

The gut microbiome plays a critical role in the health of many animals. Honeybees are no exception, as they host a core microbiome that affects their nutrition and immune function. However, the relationship between the honeybee immune system and its gut symbionts is poorly understood. Here, we explore how the beneficial symbiont Snodgrassella alvi affects honeybee immune gene expression. We show that both live and heat-killed S. alvi protect honeybees from the opportunistic pathogen Serratia marcescens and lead to the expression of host antimicrobial peptides. Honeybee immune genes respond differently to live S. alvi compared to heat-killed S. alvi, the latter causing a more extensive immune expression response. We show a preference for Toll pathway upregulation over the Imd pathway in the presence of both live and heat-killed S. alvi. Finally, we find that live S. alvi aids in clearance of S. marcescens from the honeybee gut, supporting a potential role for the symbiont in colonization resistance. Our results show that colonization by the beneficial symbiont S. alvi triggers a replicable honeybee immune response. These responses may benefit the host and the symbiont, by helping to regulate gut microbial members and preventing overgrowth or invasion by opportunists.

Engineered symbionts activate honey bee immunity and limit pathogens.

Honey bees are essential pollinators threatened by colony losses linked to the spread of parasites and pathogens. Here, we report a new approach for manipulating bee gene expression and protecting bee health. We engineered a symbiotic bee gut bacterium, Snodgrassella alvi, to induce eukaryotic RNA interference (RNAi) immune responses. We show that engineered S. alvi can stably recolonize bees and produce double-stranded RNA to activate RNAi and repress host gene expression, thereby altering bee physiology, behavior, and growth. We used this approach to improve bee survival after a viral challenge, and we show that engineered S. alvi can kill parasitic Varroa mites by triggering the mite RNAi response. This symbiont-mediated RNAi approach is a tool for studying bee functional genomics and potentially for safeguarding bee health.

Fatty-falciform ligament appendage torsion (F-FLAT): Diagnosis and management in a pediatric patient.

Fatty-falciform ligament appendage torsion (F-FLAT) is a rare condition manifested by torsion of the extraperiotoneal fat within the falciform ligament. It is similar to intraperitoneal focal fatty infarctions, including omental infarction and epiploic appendagitis. We report herein the first case of F-FLAT in a pediatric patient that failed conservative management. Ultrasound and CT scan facilitated prompt diagnosis of this rare finding and expedited a quick trial of conservative management. Despite conservative treatment, symptoms persisted for 4 weeks, but resolved after laparoscopic single-site surgical resection. In this report, we discuss the clinical features, key radiographic findings, and treatment options for this unique condition. This is only the third reported pediatric case of F-FLAT in the literature, and the first pediatric case to require surgical resection.

Case Report on Fear of Falling Syndrome: A Debilitating but Curable Gait Disorder.

BACKGROUND Fear of falling syndrome is a rare and often-missed disorder among patients with new-onset gait abnormality. The disorder is often mistaken for an organic neurologic problem, with some considering it to be a medical emergency. CASE REPORT This case report presents a 70-year-old man who presented to the Emergency Department due to inability to rise from a chair or ambulate independently. Onset of his chief complaint occurred subsequent to a traumatic fall in a public location. He underwent extensive workup, and an organic neurologic cause was ruled out. He was subsequently diagnosed with fear of falling syndrome after obtaining a detailed fall history, as well as utilizing a verified survey (the Falls Efficacy Scale-International). After extensive inpatient treatment, the patient improved significantly. However, upon discharge to a skilled nursing facility, he was not offered the recommended treatment. When he was readmitted for an unrelated reason 3 months later, he had regressed to the state he was in at the time of prior admission. CONCLUSIONS This case presents a rare debilitating but reversible gait disorder, and highlights the importance of assessing "fall history" and fear of falling in older adults. Uniquely, this case presents the rapid fluctuation in outcomes dependent on treatment, and what happens when a patient fails to complete treatment regimens. The report also provides an overview of fear of falling with the corresponding gait disorder.