The RNA Virome and Its Dynamics in an Invasive Fruit Fly, Bactrocera dorsalis, Imply Interactions Between Host and Viruses.

The oriental fruit fly, Bactrocera dorsalis (Hendel), is an important invasive agricultural insect pest with a wide host range, and has spread around the world over the last century. This evolutionary trait may have arisen primarily from interactions between B. dorsalis and other invertebrates that share the same ecological niches. The invasive behavior of B. dorsalis also frequently exposes them to diverse species of viruses. Thereby, RNA viromes may be useful microbial markers to understand the ecological evolution of B. dorsalis as well as to investigate virus-host interactions. Here, we reported eight novel RNA viruses in B. dorsalis of a lab colony, including four positive-strand RNA viruses, two negative-strand RNA viruses, and two double-stranded RNA viruses using high-throughput sequencing technology. Analysis of the virus-derived small RNAs suggested that most of these viruses may be active and trigger the host antiviral RNAi responses. The viruses were also detected in various geographical populations of B. dorsalis, implying that there is a strong association between the viromes and host. In addition, these viruses infected specific fly tissues, predominately the central nervous system and gut. Furthermore, we explored the dynamics of the viruses when hosts were exposed to short- or long-term stressors, which showed that titers of some viruses were responsively altered in the stressed B. dorsalis. The discovery of these viruses may enrich our understanding of the species diversity of RNA viruses and also provide information on viruses in association with host adaptation in insects.

Tyrosine hydroxylase coordinates larval-pupal tanning and immunity in oriental fruit fly (Bactrocera dorsalis).

BACKGROUND: The oriental fruit fly Bactrocera dorsalis (Hendel), a notorious world pest infesting fruits and vegetables, has evolved a high level of resistance to many commonly used insecticides. In this study, we investigate whether tyrosine hydroxylase (TH) that is required for cuticle tanning (sclerotization and pigmentation) in many insects, could be a potential target in controlling B. dorsalis. RESULTS: We cloned TH cDNA (BdTH) of B. dorsalis. The complete open reading frame of BdTH (KY911196) was 1737 bp in length, encoding a protein of 578 amino acids. Quantitative real-time PCR confirmed that BdTH was highly expressed in the epidermis of 3rd instar larvae, and its expression increased prior to pupation, suggesting a role in larval-pupal cuticle tanning. When we injected dsBdTH or 3-iodo-tyrosine (3-IT) as a TH inhibitor or fed insect diet supplemented with 3-IT, there was significant impairment of larval-pupal cuticle tanning and a severe obstacle to eclosion in adults followed by death in most. Furthermore, injection of Escherichia coli into larvae fed 3-IT resulted in 92% mortality and the expressions of four antimicrobial peptide genes were significantly downregulated. CONCLUSION: These results suggest that BdTH might play a critical role in larval-pupal tanning and immunity of B. dorsalis, and could be used as a potential novel target for pest control. © 2017 Society of Chemical Industry.

Blue light-induced immunosuppression in Bactrocera dorsalis adults, as a carryover effect of larval exposure.

Detrimental effects of ultraviolet (UV) light on living organisms are well understood, little is known about the effects of blue light irradiation. Although a recent study revealed that blue light caused more harmful effects on insects than UV light and blue light irradiation killed insect pests of various orders including Diptera, the effects of blue light on physiology of insects are still largely unknown. Here we studied the effects of blue light irradiation on cuticular melanin in larval and the immune response in adult stage of Bactrocera dorsalis. We also evaluated the effects of blue light exposure in larval stage on various age and mass at metamorphosis and the mediatory role of cuticular melanin in carryover effects of larval stressors across metamorphosis. We found that larvae exposed to blue light decreased melanin contents in their exoskeleton with smaller mass and delayed metamorphosis than insects reared without blue light exposure. Across metamorphosis, lower melanotic encapsulation response and higher susceptibility to Beauveria bassiana was detected in adults that had been exposed to blue light at their larval stage, thereby constituting the first evidence that blue light impaired adult immune function in B. dorsalis as a carryover effect of larval exposure.

The PLA2 gene mediates the humoral immune responses in Bactrocera dorsalis (Hendel).

The phospholipase A2 (PLA2) gene encodes the enzyme that catalyzes the hydrolysis of phospholipids (PLs) from the sn-2 position. However, little is known about its role in humoral immune responses. In this study, we investigated the expression profile of PLA2 in different tissues and developmental stages in Bactrocera dorsalis (Hendel), and the results showed that the transcriptional level of PLA2 was high in the egg and mature stage and in the testis tissue. Bacterial infection increased the expression of PLA2, and the highest degree of up-regulation appeared in the fat body. Silencing PLA2 influenced the expression of immune-related genes, including MyD88 and defensin in the Toll pathway and relish and diptericin in the Imd pathway. Moreover, the expression of MyD88 and defensin was down-regulated significantly in the ds-PLA2 group compared with those in the ds-egfp group when B. dorsalis was infected with L. monocytogenes and S. aureus, indicating that PLA2 was involved in the activation of the Toll pathway. Meanwhile, infection with L. monocytogenes and E. coli, which activate the Imd pathway, does not increase the mRNA levels of relish and diptericin in the ds-PLA2 group as severely as it increases those in the ds-egfp group, indicating that the Imd pathway was also repressed after silencing PLA2. Notably, the development of lipid droplets in fat body cells was influenced by silencing PLA2, implying that PLA2 affects the function of fat body tissue. These results suggest that the PLA2 gene may mediate humoral immune responses by reducing lipid storage in fat body cells in B. dorsalis.

Functional analysis of a NF-κB transcription factor in the immune defense of Oriental fruit fly, Bactrocera dorsalis Hendel (Diptera: Tephritidae).

Although some novel antimicrobial peptides (AMP) have been successfully isolated from Bactrocera dorsalis Hendel, the mechanisms underlying the induction of these peptides are still elusive. The homolog of NF-κB transcription factor Relish, designated as BdRelish, was cloned from B. dorsalis. The full length cDNA of BdRelish is 3954 bp with an open reading frame that encodes 1013 amino acids. Similar to Drosophila Relish and the mammalian p100, it is a compound protein containing a conserved Rel homology domain, an IPT (Ig-like, plexins, transcription factors) domain and an IκB-like domain (four ankyrin repeats), the nuclear localization signal RKRRR is also detected at the residues 449-453, suggesting that it has homology to Relish and it is a member of the Rel family of transcription activator proteins. Reverse transcription quantitative polymerase chain reaction analysis reveals that BdRelish mRNAs are detected in different quantities from various tissues and the highest transcription level of BdRelish is determined in fat body. The injection challenge of Escherichia coli and Staphylococcus aureas significantly upregulated the expression of BdRelish. The injection of BdRelish dsRNA markedly reduced the expression of BdRelish and decreased the transcription magnitude of antimicrobial peptides. Individuals injected BdRelish dsRNA died at a significantly faster rate compared with the control groups. Therefore, BdRelish is vital for the transcription of AMPs to attack the invading bacteria.

The noa gene is functionally linked to the activation of the Toll/Imd signaling pathways in Bactrocera dorsalis (Hendel).

The noa gene is an essential gene encoding a very long chain fatty acid elongase. In this study, we cloned the noa gene of Bactrocera dorsalis, which encodes a protein sharing 84.50% identity to the NOA in Drosophila melanogaster. The expression profiles indicated that the transcriptional level of noa was high at the egg stage and in the testis tissue. The results showed that noa expression was up-regulated after Listeria monocytogenes, Staphylococcus aureus and Escherichia coli infection. Silencing of noa would influence the expression of immune related genes, including MyD88 and defensin in the Toll pathway and relish and diptericin in the Imd pathway. Moreover, infection with L. monocytogenes and S. aureus after feeding ds-noa, the expression of MyD88 and defensin down-regulated significantly in ds-noa group compared with in ds-egfp group, indicating that noa interference influenced the activation of the Toll pathway. Meanwhile, infection with L. monocytogenes and E. coli, which activated the Imd pathway, do not cause increase of the mRNA levels of relish and diptericin in ds-noa group as severely as in ds-egfp treatment, indicating that the Imd pathway was also repressed after silences of noa.

Transcriptome analysis to identify genes for peptides and proteins involved in immunity and reproduction from male accessory glands and ejaculatory duct of Bactrocera dorsalis.

In the male reproductive system of insects, the male accessory glands and ejaculatory duct (MAG/ED) are important organs and their primary function is to enhance the fertility of spermatozoa. Proteins secreted by the MAG/ED are also known to induce post-mating changes and immunity responses in the female insect. To understand the gene expression profile in the MAG/ED of the oriental fruit fly Bactrocera dorsalis (Hendel), that is an important pest in fruits, we performed an Illumina-based deep sequencing of mRNA. This yielded 54,577,630 clean reads corresponding to 4.91Gb total nucleotides that were assembled and clustered to 30,669 unigenes (average 645bp). Among them, 20,419 unigenes were functionally annotated to known proteins/peptides in Gene Orthology, Clusters of Orthologous Groups, Kyoto Encyclopedia of Genes and Genomes pathway databases. Typically, many genes were involved in immunity and these included microbial recognition proteins and antimicrobial peptides. Subsequently, the inducible expression of these immunity-related genes was confirmed by qRT-PCR analysis when insects were challenged with immunity-inducible factors, suggesting their function in guaranteeing fertilization success. Besides, we identified some important reproductive genes such as juvenile hormone- and ecdysteroid-related genes in this de novo assembly. In conclusion, this transcriptomic sequencing of B. dorsalis MAG/ED provides insights to facilitate further functional research of reproduction, immunity and molecular evolution of reproductive proteins in this important agricultural pest.

Humoral immunocompetence shifts in response to developmental stage change and mating access in Bactrocera dorsalis Hendel (Diptera: Tephritidae).

Because immune defenses are often costly employed, insect immunocompetence cannot be always maintained at its maximum level. Here, the oriental fruit fly, Bactrocera dorsalis (Hendel), was used as a study object to investigate how its immune defenses varied with the developmental stage change and mating access. Our data indicated that both phenoloxidase (PO) activity and antibacterial activity significantly increased from new larvae to pupae but decreased in adults after emergence. Furthermore, both the PO activity and antibacterial activity in the hemolymph of copulated male and female adults were dramatically higher than that of virgin male and female ones, respectively. It provided the evidence that copulation could increase the magnitude of immune defense in hemolymph of B. dorsalis. Together, these results suggest that B. dorsalis possess a flexible investment strategy in immunity to meet its specific needs based on the endo- and exogenous factors, such as their distinct food source and living environments.

De novo cloning and annotation of genes associated with immunity, detoxification and energy metabolism from the fat body of the oriental fruit fly, Bactrocera dorsalis.

The oriental fruit fly, Bactrocera dorsalis, is a destructive pest in tropical and subtropical areas. In this study, we performed transcriptome-wide analysis of the fat body of B. dorsalis and obtained more than 59 million sequencing reads, which were assembled into 27,787 unigenes with an average length of 591 bp. Among them, 17,442 (62.8%) unigenes matched known proteins in the NCBI database. The assembled sequences were further annotated with gene ontology, cluster of orthologous group terms, and Kyoto encyclopedia of genes and genomes. In depth analysis was performed to identify genes putatively involved in immunity, detoxification, and energy metabolism. Many new genes were identified including serpins, peptidoglycan recognition proteins and defensins, which were potentially linked to immune defense. Many detoxification genes were identified, including cytochrome P450s, glutathione S-transferases and ATP-binding cassette (ABC) transporters. Many new transcripts possibly involved in energy metabolism, including fatty acid desaturases, lipases, alpha amylases, and trehalose-6-phosphate synthases, were identified. Moreover, we randomly selected some genes to examine their expression patterns in different tissues by quantitative real-time PCR, which indicated that some genes exhibited fat body-specific expression in B. dorsalis. The identification of a numerous transcripts in the fat body of B. dorsalis laid the foundation for future studies on the functions of these genes.

Variability of venom components in immune suppressive parasitoid wasps: from a phylogenetic to a population approach.

Endoparasitoid wasps develop at the expense of other insects, leading to their death. Eggs deposited inside the host body induce an immune response, which results in the formation of a melanized cellular capsule around the egg. To evade or counteract this response, endoparasitoids have evolved different strategies, the most often reported being injection into the host of immunosuppressive factors, notably venom proteins, along with the egg. The analysis of venom components has been performed independently in species of different taxa, but the present picture is far from complete. Intriguingly, the question of the level of venom variability inside species has been neglected, although it may partly determine the potential for parasitoid adaptation. Here, we present a short review of our present knowledge of venom components in endoparasitoids, as well as of the only well-known example of intraspecific variability in a venom immune suppressive protein being responsible for variation in parasitoid virulence. We then present data evidencing inter-individual variation of venom protein profiles, using a gel electrophoresis approach, both in laboratory strains and field populations of a figitid and a braconid species. Whether occurrence of such variability may permit a selection of parasitoid venom components driven by the host remains to be tested, notably in the context of the production and use of biological control auxiliaries.

Expression of the rubber-like protein, resilin, in developing and functional insect cuticle determined using a Drosophila anti-Rec 1 resilin antibody.

BACKGROUND: The natural elastomeric protein, insect resilin, is the most efficient elastic material known, used to store energy for jumping and flight in a variety of insects. Here, an antibody to recombinant Drosophila melanogaster pro-resilin is used to examine resilin expression in Drosophila and a wider range of insects. RESULTS: Immunostaining of Drosophila embryos reveals anti-resilin reactivity in epidermal patches that exhibit a dynamic spatial and temporal expression through late embryogenesis. Resilin is also detected in stretch receptors in the embryo. In developing adult Drosophila, resilin pads are described at the base of wings and at the base of flexible sensory hairs in pupae. Resilin is also detected in embryos of the tephritid fruitfly, Bactrocera tryoni, and two well-known concentrations of insect resilin: the flight muscle tendon of the dragonfly and the pleural arch of the flea. CONCLUSIONS: The anti-Rec1 antibody antibody developed using Drosophila pro-resilin as antigen is cross-reactive and is useful for detection of resilin in diverse insects. For the first time, resilin expression has been detected during embryogenesis, revealing segmental patches of resilin in the developing epidermis of Drosophila.