Rickettsia symbionts spread via mixed mode transmission, increasing female fecundity and sex ratio shift by host hormone modulating.

Heritable symbionts are common among animals in nature, but the molecular mechanisms underpinning symbiont invasions of host populations have been elusive. In this study, we demonstrate the spread of Rickettsia in an invasive agricultural pest, the whitefly Bemisia tabaci Mediterranean (MED), across northeastern China from 2018 to 2023. Here, we show that the beneficial symbiont Rickettsia spreads by manipulating host hormone signals. Our analyses suggest that Rickettsia have been horizontally acquired by B. tabaci MED from another invasive whitefly B. tabaci Middle East-Asia Minor 1 during periods of coexistence. Rickettsia is transmitted maternally and horizontally from female B. tabaci MED individuals. Rickettsia infection enhances fecundity and results in female bias among whiteflies. Our findings reveal that Rickettsia infection stimulates juvenile hormone (JH) synthesis, in turn enhancing fecundity, copulation events, and the female ratio of the offspring. Consequently, Rickettsia infection results in increased whitefly fecundity and female bias by modulating the JH pathway. More female progeny facilitates the transmission of Rickettsia. This study illustrates that the spread of Rickettsia among invasive whiteflies in northeastern China is propelled by host hormone regulation. Such symbiont invasions lead to rapid physiological and molecular evolution in the host, influencing the biology and ecology of an invasive species.

Insect Bacteriocytes: Adaptation, Development, and Evolution.

Bacteriocytes are host cells specialized to harbor symbionts in certain insect taxa. The adaptation, development, and evolution of bacteriocytes underlie insect symbiosis maintenance. Bacteriocytes carry enriched host genes of insect and bacterial origin whose transcription can be regulated by microRNAs, which are involved in host-symbiont metabolic interactions. Recognition proteins of peptidoglycan, the bacterial cell wall component, and autophagy regulate symbiont abundance in bacteriocytes. Horizontally transferred genes expressed in bacteriocytes influence the metabolism of symbiont peptidoglycan, which may affect the bacteriocyte immune response against symbionts. Bacteriocytes release or transport symbionts into ovaries for symbiont vertical transmission. Bacteriocyte development and death, regulated by transcriptional factors, are variable in different insect species. The evolutionary origin of insect bacteriocytes remains unclear. Future research should elucidate bacteriocyte cell biology, the molecular interplay between bacteriocyte metabolic and immune functions, the genetic basis of bacteriocyte origin, and the coordination between bacteriocyte function and host biology in diverse symbioses.

Lysine provisioning by horizontally acquired genes promotes mutual dependence between whitefly and two intracellular symbionts.

Horizontal gene transfer is widespread in insects bearing intracellular symbionts. Horizontally transferred genes (HTGs) are presumably involved in amino acid synthesis in sternorrhynchan insects. However, their role in insect-symbiont interactions remains largely unknown. We found symbionts Portiera, Hamiltonella and Rickettsia possess most genes involved in lysine synthesis in the whitefly Bemisia tabaci MEAM1 although their genomes are reduced. Hamiltonella maintains a nearly complete lysine synthesis pathway. In contrast, Portiera and Rickettsia require the complementation of whitefly HTGs for lysine synthesis and have lysE, encoding a lysine exporter. Furthermore, each horizontally transferred lysine gene of ten B. tabaci cryptic species shares an evolutionary origin. We demonstrated that Hamiltonella did not alter the titers of Portiera and Rickettsia or lysine gene expression of Portiera, Rickettsia and whiteflies. Hamiltonella also did not impact on lysine levels or protein localization in bacteriocytes harboring Portiera and ovaries infected with Rickettsia. Complementation with whitefly lysine synthesis HTGs rescued E. coli lysine gene knockout mutants. Silencing whitefly lysA in whiteflies harboring Hamiltonella reduced lysine levels, adult fecundity and titers of Portiera and Rickettsia without influencing the expression of Hamiltonella lysA. Furthermore, silencing whitefly lysA in whiteflies lacking Hamiltonella reduced lysine levels, adult fecundity and titers of Portiera and Rickettsia in ovarioles. Therefore, we, for the first time, demonstrated an essential amino acid lysine synthesized through HTGs is important for whitefly reproduction and fitness of both obligate and facultative symbionts, and it illustrates the mutual dependence between whitefly and its two symbionts. Collectively, this study reveals that acquisition of horizontally transferred lysine genes contributes to coadaptation and coevolution between B. tabaci and its symbionts.

H55 N variation in citrate synthase leads to decrement in the enzyme activity and transport rate to mitochondria in HEI-OC1 cells.

A/J mouse is a typical animal model of age-related deafness. Previous studies have shown that the mice suffer from progressive hearing loss and degeneration of cochlear cells, and a variation of H55 N in citrate synthase (CS) causes about 40% the hearing loss. CS is a key enzyme in the tricarboxylic acid cycle, which is transported from cytoplasm to mitochondria after synthesis, sorted by the mitochondrial targeting sequence (MTS). To explore the mechanism of CS (H55 N) variation in affecting its function, HEI-OC1 cells were infected with lentivirus particles to express CS-Flag or CS(H55 N)-Flag. The results showed that H55 N variation in CS, as purified by co-immunoprecipitation, decreased the enzyme activity by about 50%. Confocal microscope co-localization indicated that the CS (H55 N) variation led to a decrement in its mitochondrial content. Western blot also showed the amount of CS(H55 N)-Flag was more than that of CS(WT)-Flag in the cytosol. The results suggest H55 N variation in CS lead to decrement of its enzyme activity and targeting transport to mitochondria. We therefore conclude that decrement in CS activity and mitochondrial delivery contributes to the degeneration of cochlear cells and thus the hearing loss in A/J mice.

Autophagy Regulates Whitefly-Symbiont Metabolic Interactions.

Nutritional symbionts are restricted to specialized host cells called bacteriocytes in various insect orders. These symbionts can provide essential nutrients to the host. However, the cellular mechanisms underlying the regulation of these insect-symbiont metabolic associations remain largely unclear. The whitefly Bemisia tabaci MEAM1 hosts "Candidatus Portiera aleyrodidarum" (here, "Ca. Portiera") and "Candidatus Hamiltonella defensa" (here, "Ca. Hamiltonella") bacteria in the same bacteriocyte. In this study, the induction of autophagy by chemical treatment and gene silencing decreased symbiont titers and essential amino acid (EAA) and B vitamin contents. In contrast, the repression of autophagy in bacteriocytes via Atg8 silencing increased symbiont titers, and amino acid and B vitamin contents. Furthermore, dietary supplementation with non-EAAs or B vitamins alleviated autophagy in whitefly bacteriocytes, elevated TOR (target of rapamycin) expression, and increased symbiont titers. TOR silencing restored symbiont titers in whiteflies after dietary supplementation with B vitamins. These data suggest that "Ca. Portiera" and "Ca. Hamiltonella" evade autophagy of the whitefly bacteriocytes by activating the TOR pathway via providing essential nutrients. Taken together, we demonstrate that autophagy plays a critical role in regulating the metabolic interactions between the whitefly and two intracellular symbionts. Therefore, this study reveals that autophagy is an important cellular basis for bacteriocyte evolution and symbiosis persistence in whiteflies. The whitefly symbiosis unravels the interactions between cellular and metabolic functions of bacteriocytes. IMPORTANCE Nutritional symbionts, which are restricted to specialized host cells called bacteriocytes, can provide essential nutrients for many hosts. However, the cellular mechanisms of regulation of animal-symbiont metabolic associations have been largely unexplored. Here, using the whitefly-"Ca. Portiera"/"Ca. Hamiltonella" endosymbiosis, we demonstrate autophagy regulates the symbiont titers and thereby alters the essential amino acid and B vitamin contents. For persistence in the whitefly bacteriocytes, "Ca. Portiera" and "Ca. Hamiltonella" alleviate autophagy by activating the TOR (target of rapamycin) pathway through providing essential nutrients. Therefore, we demonstrate that autophagy plays a critical role in regulating the metabolic interactions between the whitefly and two intracellular symbionts. This study also provides insight into the cellular basis of bacteriocyte evolution and symbiosis persistence in the whitefly. The mechanisms underlying the role of autophagy in whitefly symbiosis could be widespread in many insect nutritional symbioses. These findings provide a new avenue for whitefly control via regulating autophagy in the future.

A novel microRNA regulates cooperation between symbiont and a laterally acquired gene in the regulation of pantothenate biosynthesis within Bemisia tabaci whiteflies.

Horizontally transferred genes (HTGs) play a key role in animal symbiosis, and some horizontally transferred genes or proteins are highly expressed in specialized host cells (bacteriocytes). However, it is not clear how HTGs are regulated, but microRNAs (miRNAs) are prime candidates given their previously demonstrated roles in symbiosis and impacts on the expression of host genes. A horizontally acquired PanBC that is highly expressed in whitefly bacteriocytes can cooperate with an obligate symbiont Portiera for pantothenate production, facilitating whitefly performance and Portiera titre. Here, we found that a whitefly miRNA, novel-m0780-5p, was up-regulated and its target panBC was down-regulated in Portiera-eliminated whiteflies. This miRNA was located in the cytoplasmic region of whitefly bacteriocytes. Injection of novel-m0780-5p agomir reduced the expression of PanBC in whitefly bacteriocytes, while injection of novel-m0780-5p antagomir enhanced PanBC expression. Agomir injection also reduced the pantothenate level, Portiera titre and whitefly performance. Supplementation with pantothenate restored Portiera titre and the fitness of agomir-injected whiteflies. Thus, we demonstrate that a whitefly miRNA regulates panBC-mediated host-symbiont collaboration required for pantothenate synthesis, benefiting the whitefly-Portiera symbiosis. Both panBC and novel-m0780-5p are present in the genomes of six Bemisia tabaci species. The expression of a novel miRNA in multiple B. tabaci species suggests that the miRNA evolved after panBC acquisition, and allowed this gene to be more tightly regulated. Our discovery provides the first account of a HTG being regulated by a miRNA from the host genome, and suggests key roles for interactions between miRNAs and HTGs in the functioning of symbiosis.

Effect of downregulated citrate synthase on oxidative phosphorylation signaling pathway in HEI-OC1 cells.

BACKGROUND: Citrate Synthase (Cs) gene mutation (locus ahL4) has been found to play an important role in progressive hearing loss of A/J mice. HEI-OC1 cells have been widely used as an in vitro system to study cellular and molecular mechanisms related to hearing lose. We previously reported the increased apoptosis and the accumulation of reactive oxygen species in shRNACs-1429 cells, a Cs low-expressed cell model from HEI-OCI. The details of the mechanism of ROS production and apoptosis mediated by the abnormal expression of Cs needed to research furtherly. METHODS: iTRAQ proteomics was utilized to detect the differentially expressed proteins (DEPs) caused by low expression of Cs. The GO and KEGG pathways analysis were performed for annotation of the differentially expressed proteins. Protein-protein interaction network was constructed by STRING online database. Immunoblotting was utilized to confirm the protein levels of the the differentially expressed proteins. RESULTS: The differentially expressed proteins were significantly enriched in various signaling pathways mainly related to mitochondrial dysfunction diseases including Parkinson's disease, Alzheimer's disease, Huntington's disease, et al. Most noteworthy, the oxidative phosphorylation pathway was most significantly suppressed in the shRNACs-1429 cells,, in which a total of 10 differentially expressed proteins were enriched and were all downregulated by the abnormal expression of Cs. The downregulations of Ndufb5, Ndufv1 and Uqcrb were confirmed by immunoblotting. Meanwhile, the ATP levels of shRNACs-1429 cells were also reduced. CONCLUSIONS: These results suggest that low level expression of Cs induces the inhibition of oxidative phosphorylation pathway, which is responsible for the high level production of reactive oxygen species and low level of ATP, leading to the apoptosis of cochlear cells. This study may provide new theories for understanding and therapy of progressive hearing loss.

The inherited bacterial symbiont Hamiltonella influences the sex ratio of an insect host.

In many intracellular symbioses, the microbial symbionts provide nutrients advantageous to the host. However, the function of Hamiltonella defensa, a symbiotic bacterium localized in specialized host cells (bacteriocytes) of a whitefly Bemisia tabaci, is uncertain. We eliminate this bacterium from its whitefly host by two alternative methods: heat treatment and antibiotics. The sex ratio of the host progeny and subsequent generations of Hamiltonella-free females was skewed from 1 : 1 (male : female) to an excess of males, often exceeding a ratio of 20 : 1. B. tabaci is haplodiploid, with diploid females derived from fertilized eggs and haploid males from unfertilized eggs. The Hamiltonella status of the insect did not affect copulation frequency or sperm reserve in the spermathecae, indicating that the male-biased sex ratio is unlikely due to the limitation of sperm but likely to be associated with events subsequent to sperm transfer to the female insects, such as failure in fertilization. The host reproductive response to Hamiltonella elimination is consistent with two alternative processes: adaptive shift in sex allocation by females and a constitutive compensatory response of the insect to Hamiltonella-mediated manipulation. Our findings suggest that a bacteriocyte symbiont influences the reproductive output of female progeny in a haplodiploid insect.

RGC-32 (Response Gene to Complement 32) Deficiency Protects Endothelial Cells From Inflammation and Attenuates Atherosclerosis.

OBJECTIVE: The objective of this study is to determine the role and underlying mechanisms of RGC-32 (response gene to complement 32 protein) in atherogenesis. APPROACH AND RESULTS: RGC-32 was mainly expressed in endothelial cells of atherosclerotic lesions in both ApoE-/- (apolipoprotein E deficient) mice and human patients. Rgc-32 deficiency (Rgc32-/-) attenuated the high-fat diet-induced and spontaneously developed atherosclerotic lesions in ApoE-/- mice without affecting serum cholesterol concentration. Rgc32-/- seemed to decrease the macrophage content without altering collagen and smooth muscle contents or lesional macrophage proliferation in the lesions. Transplantation of WT (wild type) mouse bone marrow to lethally irradiated Rgc32-/- mice did not alter Rgc32-/--caused reduction of lesion formation and macrophage accumulation, suggesting that RGC-32 in resident vascular cells, but not the macrophages, plays a critical role in the atherogenesis. Of importance, Rgc32-/- decreased the expression of ICAM-1 (intercellular adhesion molecule-1) and VCAM-1 (vascular cell adhesion molecule-1) in endothelial cells both in vivo and in vitro, resulting in a decrease in TNF-α (tumor necrosis factor-α)-induced monocyte-endothelial cell interaction. Mechanistically, RGC-32 mediated the ICAM-1 and VCAM-1 expression, at least partially, through NF (nuclear factor)-κB signaling pathway. RGC-32 directly interacted with NF-κB and facilitated its nuclear translocation and enhanced TNF-α-induced NF-κB binding to ICAM-1 and VCAM-1 promoters. CONCLUSIONS: RGC-32 mediates atherogenesis by facilitating monocyte-endothelial cell interaction via the induction of endothelial ICAM-1 and VCAM-1 expression, at least partially, through NF-κB signaling pathway.

RGC-32 Deficiency Protects against Hepatic Steatosis by Reducing Lipogenesis.

Hepatic steatosis is associated with insulin resistance and metabolic syndrome because of increased hepatic triglyceride content. We have reported previously that deficiency of response gene to complement 32 (RGC-32) prevents high-fat diet (HFD)-induced obesity and insulin resistance in mice. This study was conducted to determine the role of RGC-32 in the regulation of hepatic steatosis. We observed that hepatic RGC-32 was induced dramatically by both HFD challenge and ethanol administration. RGC-32 knockout (RGC32(-/-)) mice were resistant to HFD- and ethanol-induced hepatic steatosis. The hepatic triglyceride content of RGC32(-/-) mice was decreased significantly compared with WT controls even under normal chow conditions. Moreover, RGC-32 deficiency decreased the expression of lipogenesis-related genes, sterol regulatory element binding protein 1c (SREBP-1c), fatty acid synthase, and stearoyl-CoA desaturase 1 (SCD1). RGC-32 deficiency also decreased SCD1 activity, as indicated by decreased desaturase indices of the liver and serum. Mechanistically, insulin and ethanol induced RGC-32 expression through the NF-κB signaling pathway, which, in turn, increased SCD1 expression in a SREBP-1c-dependent manner. RGC-32 also promoted SREBP-1c expression through activating liver X receptor. These results demonstrate that RGC-32 contributes to the development of hepatic steatosis by facilitating de novo lipogenesis through activating liver X receptor, leading to the induction of SREBP-1c and its target genes. Therefore, RGC-32 may be a potential novel drug target for the treatment of hepatic steatosis and its related diseases.