PAP1 activates the prophenoloxidase system against bacterial infection in Musca domestica.

We previously identified three putative prophenoloxidase-activating proteinase (mdPAP1, mdPAP2, and mdPAP3) genes from housefly Musca domestica by transcriptomic analysis. In this study, mdPAP1 cDNA was cloned, and the function of its encoded protein was analyzed. The cDNA of mdPAP1 was 1358 bp, and it contained a single open reading frame of 1122 bp encoding a predicted MdPAP1 protein of 373 amino acids. The estimated molecular weight of MdPAP1 was 41267.08 Da with an isoelectric point of 6.25. The deduced amino acid sequence of MdPAP1 exhibited high similarity to known PAPs of insects. mdPAP1 was detected in larvae, pupae, and adult housefly, and the expression level of mdPAP1 was upregulated in bacterial challenged larvae. The recombinant protein of MdPAP1 expressed in Escherichia coli could cleave the prophenoloxidase into phenoloxidase in M. domestica hemolymph infected by bacteria and result in a significant increase of the total phenoloxidase activity. In addition, RNA interference-mediated gene silencing of mdPAP1 significantly increased the mortality of M. domestica larvae. Results indicated that mdPAP1 was involved in the activation of the prophenoloxidase against bacterial infection in M. domestica.

Identifying and Characterizing a Novel Peritrophic Matrix Protein (MdPM-17) Associated With Antibacterial Response From the Housefly, Musca domestica (Diptera: Muscidae).

Peritrophic matrix/membrane (PM) critically prevents the midgut of insects from external invasion by microbes. The proteins in the peritrophic membrane are its major structural components. Additionally, they determine the formation and function of this membrane. However, the role of PM proteins in immune regulation is unclear. Herein, we isolated a novel PM protein (MdPM-17) from Musca domestica larvae. Further, the function of MdPM-17 in regulating host innate immunity was identified. Results showed that the cDNA of MdPM-17 full is 635 bp in length. Moreover, it consists of a 477-bp open reading frame encoding 158 amino acid residues. These amino acid residues are composed of two Chitin-binding type-2 domain (ChtBD2) and 19 amino acids as a signal peptide. Moreover, tissue distribution analysis indicates that MdPM-17 was enriched expressed in midgut, and moderate levels in the fat body, foregut, and malpighian tubule. Notably, MdPM-17 recombinant protein showed high chitin-binding capacity, thus belongs to the Class III PM protein group. MdPM-17 protein silencing via RNA interference resulted in the expression of antimicrobial peptide (defensin, cecropins, and diptericin) genes, and this occurred after oral inoculation with exogenous microbes Escherichia coli (Enterobacteriales:Enterobacteriaceae), Staphylococcus aureus (Bacillales:Staphylococcaceae), and Candida albicans (Endomycetales:Saccharomycetaceae)). Therefore, all the antimicrobial peptide (AMP) gene expression levels are high in MdPM-17-depleted larvae during microbial infection compared to controls. Consequently, these findings indicate that MdPM-17 protein is associated with the antibacterial response from the housefly.

Two ferritin genes (MdFerH and MdFerL) are involved in iron homeostasis, antioxidation and immune defense in housefly Musca domestica.

Ferritin is a ubiquitous multi-subunit iron storage protein, made up of heavy chain and light chain subunits. In recent years, invertebrate ferritins have emerged as an important, yet largely underappreciated, component of host defense and antioxidant system. Here, two alternatively spliced transcripts encoding for a unique ferritin heavy chain homolog (MdFerH), and a transcript encoding for a light chain homolog (MdFerL) are cloned and characterized from Musca domestica. Comparing with MdFerH1, a fragment is absent at the 5' untranslated region of MdFerH2, where a putative iron response element is present. Amino acid sequence analysis shows that MdFerH possesses a strictly conserved ferroxidase site, while MdFerL has a putative atypical active center. Tissue distribution analysis indicates that MdFers are enriched expressed in gut. When the larvae receive diverse stimulations, including challenge by bacteria, exposure to excess Fe2+, doxorubicin or ultraviolet, the expression of MdFers is positively up-regulated in different degrees and different temporal patterns, indicating their potential roles in oxidative stress. The two mRNA isoforms of MdFerH appear to be differentially expressed in different tissues, but seem to show the similar expression patterns under diverse stress conditions. Further investigation reveals that silencing MdFers can alter the redox homeostasis, leading elevated mortalities of larvae following bacterial infection. Inspiringly, recombinant MdFerL produced in Pichia pastoris shows significant iron-chelating activity in vitro. These results suggest a pivotal role of ferritins from housefly in iron homeostasis, antibacterial immunity and redox balance.

Involvement of TRAF6 in regulating immune defense and ovarian development in Musca domestica.

The tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) is a key cytoplasm signaling adaptor that mediates signals activated by TNFR superfamily and the interleukin-1/Toll-like receptor (IL-1/TLR) superfamily. In the present research, a housefly Musca domestica TRAF6 (MdTRAF6) gene is identified and characterized, with a 51.7-kDa protein possessing a RING domain and a conserved C-terminal TRAF homology MATH domain encoded. MdTRAF6 is widely expressed in diverse tissues with high expression levels in gut and fat body, which is of the highest levels in adult in all growth stages. The expression of MdTRAF6 could be remarkably induced by bacterial challenge, and the silencing MdTRAF6 could alter the expressions of NF-κB-like genes (relish and dorsal) and antimicrobial peptide genes (cecropin, diptericin, attacin, muscin), thus leading elevated mortalities of larvae followed by bacterial infection. Inspiringly. MdTRAF6-depleted adult flies display higher mortality, lower fertility and reduced survival of offspring than the controls. Further investigation reveals that knockdown of MdTRAF6 disturbs the ovarian development and impaires the expressions of vitellogenin and vitellogenin receptor genes in the adult females. All these phenotypes show crucial roles of MdTRAF6 in innate immunity via positive regulation of the Toll pathway and negative regulation of the Imd pathway, and in reproduction by maintaining ovarian development.

Antimicrobial functional divergence of the cecropin antibacterial peptide gene family in Musca domestica.

BACKGROUND: It has been reported that there are more than ten antimicrobial peptides (AMPs) belonging to the cecropin family in Musca domestica; however, few of them have been identified, and the functions of the other molecules are poorly understood. METHODS: Sequences of the M. domestica cecropin family of genes were cloned from cDNA template, which was reverse-transcribed from total mRNA isolated from third-instar larvae of M. domestica that were challenged with pathogens. Sequence analysis was performed using DNAMAN comprehensive analysis software, and a molecular phylogenetic tree of the cecropin family was constructed using the Neighbour-Joining method in MEGA v.5.0 according to the mature peptide sequences. Antibacterial activity of the synthetic M. domestica cecropin protein was detected and the minimum inhibitory concentration (MIC) values were determined using broth microdilution techniques. Time-killing assays were performed on the Gram-negative bacteria, Acinetobacter baumannii, at the logarithmic or stabilizing stages of growth, and its morphological changes when treated with Cec4 were assessed by scanning electron microscopy (SEM) and detection of leakage of 260 nm absorbing material. RESULTS: Eleven cecropin family genes, namely Cec01, Cec02 and Cec1-9, show homology to the Cec form in a multigene family on the Scaffold18749 of M. domestica. In comparing the encoded cecropin protein sequences, most of them have the basic characteristics of the cecropin family, containing 19 conservative amino acid residues. To our knowledge, this is the first experimental demonstration that most genes in the Cec family are functional. Cec02, Cec1, Cec2, Cec5 and Cec7 have similar antibacterial spectra and antibacterial effects against Gram-negative bacteria, while Cec4 displays a more broad-spectrum of antimicrobial activity and has a very strong effect on A. baumannii. Cec4 eliminated A. baumannii in a rapid and concentration-dependent manner, with antibacterial effects within 24 h at 1× MIC and 2× MIC. Furthermore, SEM analysis and the leakage of 260 nm absorbing material detection indicated that Cec4 sterilized the bacteria through the disruption of cell membrane integrity. CONCLUSIONS: Although there are more than ten cecropin genes related to M. domestica, some of them have no preferred antibacterial activity other than Cec4 against A. baumannii.

A house fly TNF ortholog Eiger regulates immune defense via cooperating with Toll and Imd pathways.

In mammals, the TNF family is important inflammatory cytokines. Eiger, the invertebrate ortholog of TNF identified firstly in Drosophila, has been implicated in immune response with an unknown molecular mechanism. The present work reports a novel eiger like gene (Mdeiger) from Musca domestica. Mdeiger was significantly up-regulated upon challenge with either Escherichia coli or Staphylococcus aureus. Silencing Mdeiger led to higher mortalities of larvae post either E. coli or S. aureus infection, enhanced the expressions of attacin and diptericin, but blocked the induction of ceropin and muscin, and inhibited the activation of phenoloxidase following bacterial challenge. Meanwhile, the expression of dorsal and JNK was inhibited while that of relish was enhanced in Mdeiger-depleted larvae. We suppose that, by coordinating with the Imd, Toll and JNK pathways, Mdeiger be involved in regulating the innate immune response through controlling the capacity of phenoloxidase and the expression of antimicrobial peptide genes synergistically.

Involvement of trehalose-6-phosphate synthase in innate immunity of Musca domestica.

Trehalose-6-phosphate synthase (TPS) is responsible for synthesizing trehalose, which is prevalent in crustaceans and insects as blood-sugar. In this paper, a TPS gene from Musca domestica(MdTPS)has been cloned and characterized. MdTPS promoter was analyzed, and its transcriptional activity was verified in vitro by Sf9 cell. Quantitative RT-PCR analysis revealed that the MdTPS transcription was up-regulated following bacterial challenge by Escherichia coli or Staphylococcus aureus. Meanwhile, trehalose is accumulated in larvae upon bacterial challenge. Significantly increased mortality can be observed in MdTPS depleted (RNA interference, RNAi) larvae under bacterial infection. Interestingly, feeding trehalose led to increasing trehalose content in larvae, and the effects of RNAi targeting MdTPS on host survival against bacterial challenge was partly counteracted. Taken together, these results suggest that MdTPS acts as an inducible anti-stress gene that takes part in immune defense in M. domestica via synthesizing its product trehalose.

Coevolution of hytrosaviruses and host immune responses.

BACKGROUND: Hytrosaviruses (SGHVs; Hytrosaviridae family) are double-stranded DNA (dsDNA) viruses that cause salivary gland hypertrophy (SGH) syndrome in flies. Two structurally and functionally distinct SGHVs are recognized; Glossina pallidipes SGHV (GpSGHV) and Musca domestica SGHV (MdSGHV), that infect the hematophagous tsetse fly and the filth-feeding housefly, respectively. Genome sizes and gene contents of GpSGHV (~ 190 kb; 160-174 genes) and MdSGHV (~ 124 kb; 108 genes) may reflect an evolution with the SGHV-hosts resulting in differences in pathobiology. Whereas GpSGHV can switch from asymptomatic to symptomatic infections in response to certain unknown cues, MdSGHV solely infects symptomatically. Overt SGH characterizes the symptomatic infections of SGHVs, but whereas MdSGHV induces both nuclear and cellular hypertrophy (enlarged non-replicative cells), GpSGHV induces cellular hyperplasia (enlarged replicative cells). Compared to GpSGHV's specificity to Glossina species, MdSGHV infects other sympatric muscids. The MdSGHV-induced total shutdown of oogenesis inhibits its vertical transmission, while the GpSGHV's asymptomatic and symptomatic infections promote vertical and horizontal transmission, respectively. This paper reviews the coevolution of the SGHVs and their hosts (housefly and tsetse fly) based on phylogenetic relatedness of immune gene orthologs/paralogs and compares this with other virus-insect models. RESULTS: Whereas MdSGHV is not vertically transmitted, GpSGHV is both vertically and horizontally transmitted, and the balance between the two transmission modes may significantly influence the pathogenesis of tsetse virus. The presence and absence of bacterial symbionts (Wigglesworthia and Sodalis) in tsetse and Wolbachia in the housefly, respectively, potentially contributes to the development of SGH symptoms. Unlike MdSGHV, GpSGHV contains not only host-derived proteins, but also appears to have evolutionarily recruited cellular genes from ancestral host(s) into its genome, which, although may be nonessential for viral replication, potentially contribute to the evasion of host's immune responses. Whereas MdSGHV has evolved strategies to counteract both the housefly's RNAi and apoptotic responses, the housefly has expanded its repertoire of immune effector, modulator and melanization genes compared to the tsetse fly. CONCLUSIONS: The ecologies and life-histories of the housefly and tsetse fly may significantly influence coevolution of MdSGHV and GpSGHV with their hosts. Although there are still many unanswered questions regarding the pathogenesis of SGHVs, and the extent to which microbiota influence expression of overt SGH symptoms, SGHVs are attractive 'explorers' to elucidate the immune responses of their hosts, and the transmission modes of other large DNA viruses.

Tissue-dependent induction of antimicrobial peptide genes after body wall injury in house fly (Musca domestica) larvae.

Injury of the insect body wall, which enables environmental microorganisms to invade into insect tissues, induces innate immune responses including the induction of antimicrobial peptides (AMPs) in flies and silkworms. Here, house fly (Musca domestica) larvae and pupae were injured using a needle and the effects on the expression of genes encoding AMPs were examined. The expression of AMP genes including defensin, attacin, diptericin, and sarcotoxin II dramatically increased in both larvae and pupae after injury of the body wall, indicating that innate immune responses were induced. Furthermore, the injury-dependent expression of AMP genes was examined in larval tissues including fat bodies, hemocytes, salivary glands, and digestive tracts. Injury-dependent AMP gene expression was observed in salivary glands, hemocytes, and fat bodies, but not in digestive tracts. The degree of the transcriptional induction of each gene differed among tissues, suggesting that their expression is governed by complex regulatory machinery and that AMPs have tissue-specific functions. To further examine the properties of the AMPs, we examined the antimicrobial activities of partial synthetic peptides corresponding to portions of the predicted AMP proteins deduced from the AMP genes. A synthetic peptide exhibited antimicrobial activity, indicating that these injury-inducible genes are potential medicinal resources.

Characterization and Expression Pattern Analysis of the T-Complex Protein-1 Zeta Subunit in Musca domestica L (Diptera).

Chaperonins, belonging to the T-complex protein-1 (TCP-1) family, assist in the correct folding of nascent and misfolded proteins. It is well-known that in mammals, the zeta subunit of the TCP-1 complex (TCP-1ζ) plays a vital role in the folding and assembly of cytoskeleta proteins. This study reported for the first time the cloning, characterization and expression pattern analysis of the TCP-1ζ from Musca domestica, which was named as MdTCP-1ζ. The MdTCP-1ζ cDNA is 1,803 bp long with a 1,596 bp open reading frame that encodes a protein with 531 bp amino acids. The analysis of the transcriptional profile of MdTCP-1ζ using qRT-PCR revealed relatively high expression in the salivary glands and trachea at the tissues while among the developmental stages. The highest expression was observed only in the eggs suggesting that the MdTCP-1ζ may play a role in embryonic development. The expression of MdTCP-1ζ was also significantly induced after exposure to short-term heat shock and infection by Escherichia coli, Staphylococcus aureus, or Candida albicans. This suggested that MdTCP-1ζ may take part in the immune responses of housefly and perhaps contribute to the protection against cellular injury.

Evaluation of Beauveria bassiana infection in the hemolymph serum proteins of the housefly, Musca domestica L. (Diptera: Muscidae).

Beauveria bassiana plays a prominent role in biocontrol of houseflies, Musca domestica (L.). Thus, a deeper insight into immune response of M. domestica during B. bassiana infection was warranted to assist the production of more efficient mycoinsecticides. The present study investigates changes in protein profile of M. domestica hemolymph serum post B. bassiana infection using two-dimensional difference gel electrophoresis (2D-DIGE) followed by identification of selected proteins by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). The non-infected or control group of flies showed an expression of 54 proteins, while M. domestica infected with B. bassiana expressed a total of 68 hemolymph serum proteins. Thirty three proteins were expressed in both groups of houseflies, whereas 35 proteins were exclusively expressed in infected flies and 21 proteins were exclusively expressed in control flies. Among the 33 proteins which were expressed in both groups of houseflies, 17 proteins showed downregulation, while16 proteins were upregulated in the infected flies compared to the non-infected ones. The results from this study are expected to facilitate better understanding of insect's immune response mechanism.

Properties of induced antimicrobial activity in Musca domestica larvae.

Insects produce antimicrobial molecules that contribute to their innate immune responses to eliminate invading microorganisms. To explore the potential utility of these antimicrobial molecules, we focused on larvae of the house fly Musca domestica, which is an efficient processor of organic waste and a good resource of protein and oil for animal feeding. The induction of hemagglutinating activity, which is usually accompanied by activation of innate immune responses in fly larvae, was observed in the hemolymph following needle injury. Hemolymph collected from injured larvae demonstrated potent antimicrobial activities against both Gram-positive and Gram-negative bacteria, including Staphylococcus aureus and Pseudomonas aeruginosa. Furthermore, the antimicrobial activity was significantly retained in hemolymph after heat-treatments, suggesting that pasteurization of animal feed prepared from fly larvae would be a useful sterilization method. These observations indicate that injured Musca domestica larvae are a source of antimicrobial agents, and highlight the utility of preparing animal feed from these larvae.

Identification and characterization of a novel antimicrobial protein from the housefly Musca domestica.

Antimicrobial peptides/proteins are immune-related molecules that are widely distributed in bacteria, fungi, plants, invertebrates and higher animals. They have exhibited great potential to be developed into antimicrobial drugs. The housefly, Musca domestica, lives in a highly contaminated environment and has adapted a robust immune system against various pathogens. As an effort to search for new antimicrobial molecules in the housefly, we investigated the function of an uncharacterized gene firstly by confirming that its expression was induced by infection in M. domestica. The corresponding protein was then shown to have potent antimicrobial activity. Scanning Electron Microscopy data showed that treatment of C. albicans cells with the protein caused cell size decreasing and cell elongation. The results here suggest the protein a novel class of antimicrobial protein and provide new insights into the immunological mechanisms by which M. domestica combats invading C. albicans.

Brevibacillus laterosporus pathogenesis and local immune response regulation in the house fly midgut.

The insect midgut represents the primary site of action of the entompathogenic bacterium Brevibacillus laterosporus. While most studies on this microorganism focus on the identification and characterization of possible virulence factors and toxins, little is known about the insect immune defense mechanisms that are activated against this pathogen. In this study we have investigated the local immune response of different house fly stages to B. laterosporus at the transcriptional level, and we tested the hypothesis that an improvement in entomopathogenicity can be achieved by impairing host innate immunity. Gene expression analyses showed that immediately after spore ingestion (6-12h) both larvae and adults increased the transcription rate of immune related genes in the midgut tissues, with special regard to those encoding for the main house fly antimicrobial peptides (AMPs) (i.e., attacin, cecropin, defensin, diptericin, domesticin, muscin) and for prophenoloxydase that is normally involved in the cascade of events leading to the generation of reactive oxygen species (ROS) and other factors with antibacterial properties. In experiments evaluating the use of an immunosuppressive agent to enhance the virulence of B. laterosporus against adult house flies, a significant downregulation of the same genes was observed 12-24h after the administration of sub-lethal doses of the botanical compound azadirachtin. Consequently, a significant increase in B. laterosporus entomopathogenic action was observed when flies were preliminarily or simultaneously exposed to a sub-lethal dose of azadirachtin. These results provide an important contribution to the prospect of employing immune-impairing tools to implement pest management strategies.

Campylobacter jejuni in Musca domestica: An examination of survival and transmission potential in light of the innate immune responses of the house flies.

The house fly, Musca domestica, has been implicated as a vector of Campylobacter spp., a major cause of human disease. Little is known whether house flies serve as biological amplifying hosts or mechanical vectors for Campylobacter jejuni. We investigated the period after C. jejuni had been ingested by house flies in which viable C. jejuni colonies could be isolated from whole bodies, the vomitus and the excreta of adult M. domestica and evaluated the activation of innate immune responses of house flies to ingested C. jejuni over time. C. jejuni could be cultured from infected houseflies soon after ingestion but no countable C. jejuni colonies were observed > 24 h postingestion. We detected viable C. jejuni in house fly vomitus and excreta up to 4 h after ingestion, but no viable bacteria were detected ≥ 8 h. Suppression subtractive hybridization identified pathogen-induced gene expression in the intestinal tracts of adult house flies 4-24 h after ingesting C. jejuni. We measured the expression of immune regulatory (thor, JNK, and spheroide) and effector (cecropin, diptericin, attacin, defensing, and lysozyme) genes in C. jejuni-infected and -uninfected house flies using quantitative real time PCR. Some house fly factor, or combination of factors, eliminates C. jejuni within 24 h postingestion. Because C. jejuni is not amplified within the body of the housefly, this insect likely serves as a mechanical vector rather than as a true biological, amplifying vector for C. jejuni, and adds to our understanding of insect-pathogen interactions.

A Comparative Study of Skin Prick Test versus Serum-Specific IgE Measurement in Indian Patients with Bronchial Asthma and Allergic Rhinitis.

BACKGROUND: Skin prick testing (SPT) is the 'gold standard' in the assessment of allergic sensitivity to inhalant allergens. Serum-specific immunoglobulin E (SSIgE) measurement is a complementary test. SPT is performed with antigen extracts from India while SSIgE utilises extracts derived from European antigens. OBJECTIVE: To evaluate the performance of allergic assessment by SSIgE against cockroach, housefly and mosquito aeroallergens which are frequently implicated in driving respiratory allergies in India considering SPT as the 'gold standard'. METHODS: Twenty patients (mean age 28.5 years; range 15-50 years) diagnosed to have bronchial asthma and/or rhinitis underwent SPT. The SSIgE levels were obtained at the same visit. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of SSIgE testing were calculated using SPT as the 'gold standard'. The correlation between SPT grading and SSIgE levels was also evaluated. RESULTS: The sensitivity of SSIgE testing to each of the 3 aero-allergens was > 85%. The PPV of cockroach and mosquito SSIgE was > 85%; housefly SSIgE had PPV of 68.7%. The two tests were in agreement in 85% (cockroach), 90% (mosquito) and 55% (housefly). There was a significant correlation between the grades of SPT reactions and SSIgE levels. CONCLUSIONS: The SSIgE has higher sensitivity and PPV, but lacks specificity. Higher sensitivity with low specificity leads to increased false positive diagnosis of allergic disease. Unlike allergenic pollens, however, insect antigen extracts from different regions seem to give comparable results, and can thus, reliably be used in the evaluation of allergy.

The effect of Beauveria bassiana infection on cell mediated and humoral immune response in house fly, Musca domestica L.

Entomopathogenic fungi that manifest infections by overcoming insect's immune response could be a successful control agent for the house fly, Musca domestica L. which is a major domestic, medical, and veterinary pest. In this study, the immune response of house fly to Beauveria bassiana infection was investigated to reveal fundamental aspects of house fly hemocyte biology, such as hemocyte numbers and size, which is poorly understood. The total hemocyte counts (THCs) in B. bassiana-infected house fly showed an initial increase (from 6 to 9 h), followed by subsequent decrease (9 to 12 h) with increase in time of infection. The THCs was slightly greater in infected flies than the non-infected ones. Insight into relative hemocyte counts depicted a significant increase in prohemocyte (PR) and decrease in granulocyte (GR) in infected house flies compared to non-infected ones. The relative cell area of hemocyte cells showed a noticeable increase in PR and intermediate cells (ICs), while a considerable reduction was observed for plasmatocyte (PL) and GR. The considerable variation in relative cell number and cell area in the B. bassiana-infected house flies indicated stress development during infection. The present study highlights changes occurring during B. bassiana invasion to house fly leading to establishment of infection along with facilitation in understanding of basic hemocyte biology. The results of the study is expected to help in better understanding of house fly immune response during fungal infection, so as to assist production of more efficient mycoinsecticides for house fly control using B. bassiana.

Transcriptional response of Musca domestica larvae to bacterial infection.

The house fly Musca domestica, a cosmopolitan dipteran insect, is a significant vector for human and animal bacterial pathogens, but little is known about its immune response to these pathogens. To address this issue, we inoculated the larvae with a mixture of Escherichia coli and Staphylococcus aureus and profiled the transcriptome 6, 24, and 48 h thereafter. Many genes known to controlling innate immunity in insects were induced following infection, including genes encoding pattern recognition proteins (PGRPs), various components of the Toll and IMD signaling pathways and of the proPO-activating and redox systems, and multiple antimicrobial peptides. Interestingly, we also uncovered a large set of novel immune response genes including two broad-spectrum antimicrobial peptides (muscin and domesticin), which might have evolved to adapt to house-fly's unique ecological environments. Finally, genes mediating oxidative phosphorylation were repressed at 48 h post-infection, suggesting disruption of energy homeostasis and mitochondrial function at the late stages of infection. Collectively, our data reveal dynamic changes in gene expression following bacterial infection in the house fly, paving the way for future in-depth analysis of M. domestica's immune system.

Pseudomonas aeruginosa in Musca domestica L.: temporospatial examination of bacteria population dynamics and house fly antimicrobial responses.

House flies associate with microbes throughout their life history. Bacteria ingested by adult flies enter the alimentary canal and face a hostile environment including antimicrobial defenses. Because the outcome of this interaction impacts bacterial survival and dissemination, our primary objective was to understand the temporospatial dynamics of fly-bacteria associations. We concurrently examined the temporospatial fate of GFP-expressing Pseudomonas aeruginosa (GFP-P. aeruginosa) in the house fly alimentary canal along with antimicrobial peptide (AMP) expression. Motile, viable GFP-P. aeruginosa were found in all regions of the alimentary canal and were culturable throughout the observation period (2-24 h). A significant decrease in recoverable bacteria occurred between 2 and 12 h, followed by an increase between 12 and 24 h. qRT-PCR analysis showed expression of the AMPs cecropin, diptericin, and defensin both locally (gut) and systemically. Furthermore, mRNA of all AMPs were expressed throughout gut tissues, with some tissue-specific temporal variation. Interestingly, fluctuation in recoverable P. aeruginosa was associated with AMP protein expression in the gut (immunofluorescent signal detection), but not with mRNA (qRTPCR). In regards to vector competence, flies excreted GFP-P. aeruginosa throughout the 24 h period, serving as both reservoirs and disseminators of this bacterium. Collectively, our data show flies can harbor and disseminate P. aeruginosa, and that the interactions of fly defenses with bacteria can influence vector competence.

Staphylococcus aureus in the house fly: temporospatial fate of bacteria and expression of the antimicrobial peptide defensin.

House flies disseminate numerous species of bacteria acquired during feeding and breeding activities in microbe-rich habitats. Previous house fly surveys have detected the pathogen Staphylococcus aureus Rosenbach 1884, which causes cutaneous and septic infections in mammals, and enterotoxic food poisoning. We assessed the fate of GFP-expressing S. aureus (GFP-S. aureus) in the house fly alimentary canal with microscopy and by culture of whole flies and excreta. Furthermore, the concurrent expression of the antimicrobial peptide gene defensin was measured in the crop, proventriculus, midgut, and fat body. As soon as 4 h postingestion (PI), GFP-S. aureus were visualized as cocci or diplococci in the hindgut and rectum of flies fed approximately equal 10(5) colony forming units. Bacteria persisted up to 6 h PI but significantly decreased. Excretion of viable GFP-S. aureus peaked at 2 h PI and, although significantly less, continued up to 4 h PI. defensin was highly upregulated locally in the alimentary canal and systemically in fat body at 2, 4, and 6 h PI making this study the first to report, to our knowledge, an epithelial and systemic response to a bacterium with lysine-type peptidoglycan in flies exposed via feeding. While flies harbored S. aureus for up to 6 h PI, the highest probability of vectoring biologically relevant amounts of bacteria occurred 0-2 h PI. The combined effects of excretion, digestion and antimicrobial effectors likely contribute to loss of ingested bacteria. Nonetheless, house flies are relevant vectors for S. aureus up to 2 h PI and environmental reservoirs up to 6 h PI.