Analysis of Genes Expression of Spodoptera exigua Larvae upon AcMNPV Infection

Background

The impact of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) infection on host gene expression in Spodoptera exigua 4th instar larvae was investigated through the use of 454 sequencing-based RNA-seq of cDNA libraries developed from insects challenged with active AcMNPV or heat-inactivated AcMNPV.

Methodology/Principal Findings

By comparing the two cDNA libraries, we show that 201 host genes are significantly up-regulated and 234 genes are significantly down-regulated by active AcMNPV infection. Down-regulated host genes included genes encoding antimicrobial peptides, namely three gloverin isoforms and an attacin, indicating that the viral infection actively repressed the expression of a portion of the host immune gene repertoire. Another interesting group of down-regulated host genes included genes encoding two juvenile hormone binding proteins and a hexamerin, all of which are involved in juvenile hormone regulation. The expression of these genes was enhanced by the topical application of Juvenile Hormone III (JHIII) in the insects challenged with heat-inactivated AcMNPV. However, infection with the active virus strongly suppresses the expression of these three genes, regardless of the absence or presence of JHIII.

Conclusions/Significance

Using RNA-seq, we have identified groups of immune-regulated and juvenile hormone-regulated genes that are suppressed by infection with active AcMNPV. This information and further studies on the regulation of host gene expression by AcMNPV will provide the tools needed to enhance the utility of the virus as an effective protein expression system and as an insecticide.     

Imaginal discs--a new source of chromosomes for genome mapping of the yellow fever mosquito Aedes aegypti

Background

The mosquito Aedes aegypti is the primary global vector for dengue and yellow fever viruses. Sequencing of the Ae. aegypti genome has stimulated research in vector biology and insect genomics. However, the current genome assembly is highly fragmented with only ∼31% of the genome being assigned to chromosomes. A lack of a reliable source of chromosomes for physical mapping has been a major impediment to improving the genome assembly of Ae. aegypti.

Methodology/Principal Findings

In this study we demonstrate the utility of mitotic chromosomes from imaginal discs of 4^th instar larva for cytogenetic studies of Ae. aegypti. High numbers of mitotic divisions on each slide preparation, large sizes, and reproducible banding patterns of the individual chromosomes simplify cytogenetic procedures. Based on the banding structure of the chromosomes, we have developed idiograms for each of the three Ae. aegypti chromosomes and placed 10 BAC clones and a 18S rDNA probe to precise chromosomal positions.

Conclusion

The study identified imaginal discs of 4^th instar larva as a superior source of mitotic chromosomes for Ae. aegypti. The proposed approach allows precise mapping of DNA probes to the chromosomal positions and can be utilized for obtaining a high-quality genome assembly of the yellow fever mosquito.     

Roles of Peroxinectin in PGE2-Mediated Cellular Immunity in Spodoptera exigua

Background

Prostaglandins (PGs) mediate insect immune responses to infections and invasions. Although the presence of PGs has been confirmed in several insect species, their biosynthesis in insects remains a conundrum because orthologs of the mammalian cyclooxygenases (COXs) have not been found in the known insect genomes. PG-mediated immune reactions have been documented in the beet armyworm, Spodoptera exigua. The purpose of this research is to identify the source of PGs in S. exigua.

Principal Findings

Peroxidases (POXs) are a sister group of COX genes. Ten putative POXs (SePOX-A ∼ SePOX-J) were expressed in S. exigua. Expressions of SePOX-F and -H were induced by bacterial challenge and expressed in the hemocytes and the fat body. RNAi of each POX was performed by hemocoelic injection of their specific double-stranded RNAs. dsPOX-F or, separately, dsPOX-H, but not the other eight dsRNA constructs, specifically suppressed hemocyte-spreading behavior and nodule formation; these two reactions were also inhibited by aspirin, a COX inhibitor. PGE₂, but not arachidonic acid, treatment rescued the immunosuppression. Sequence analysis indicated that both POX genes were clustered with peroxinectin (Pxt) and their cognate proteins shared some conserved domains corresponding to the Pxt of Drosophila melanogaster.

Conclusions

SePOX-F and -H are Pxt-like genes associated with PG biosynthesis in S. exigua.     

Different Functions of the Insect Soluble and Membrane-Bound Trehalase Genes in Chitin Biosynthesis Revealed by RNA Interference

Background

Trehalase, an enzyme that hydrolyzes trehalose to yield two glucose molecules, plays a pivotal role in various physiological processes. In recent years, trehalase proteins have been purified from several insect species and are divided into soluble (Tre-1) and membrane-bound (Tre-2) trehalases. However, no functions of the two trehalases in chitin biosynthesis in insects have yet been reported.

Principal Findings

The membrane-bound trehalase of Spodoptera exigua (SeTre-2) was characterized in our laboratory previously. In this study, we cloned the soluble trehalase gene (SeTre-1) and investigated the tissue distribution and developmental expression pattern of the two trehalase genes. SeTre-1 was expressed highly in cuticle and Malpighian tubules, while SeTre-2 was expressed in tracheae and fat body. In the midgut, the two trehalase genes were expressed in different locations. Additionally, the expression profiles of both trehalase mRNAs and their enzyme activities suggest that they may play different roles in chitin biosynthesis. The RNA interference (RNAi) of either SeTre-1 or SeTre-2 was gene-specific and effective, with efficiency rates up to 83% at 72 h post injection. After RNAi of SeTre-1 and SeTre-2, significant higher mortality rates were observed during the larva-pupa stage and pupa-adult stage, and the lethal phenotypes were classified and analyzed. Additionally, the change trends of concentration of trehalose and glucose appeared reciprocally in RNAi-mutants. Moreover, knockdown of SeTre-1 gene largely inhibited the expression of chitin synthase gene A (CHSA) and reduced the chitin content in the cuticle to two-thirds relative to the control insects. The chitin synthase gene B (CHSB) expression, however, was inhibited more by the injection of dsRNA for SeTre-2, and the chitin content in the midgut decreased by about 25%.

Conclusions

SeTre-1 plays a major role in CHSA expression and chitin synthesis in the cuticle, and SeTre-2 has an important role in CHSB expression and chitin synthesis in the midgut.     

A fly in the ointment: evaluation of traditional use of plants to repel and kill blowfly larvae in fermented fish

Introduction

In rural areas in Laos, fly larvae infestations are common in fermenting fish. Blowflies (Chrysomya megacephala, Diptera: Calliphoridae) are attracted to oviposit (and/or larviposit) onto fermenting fish which results in infestations with fly larvae. Knowledge of traditional use of plants to repel larvae during the production of fermented fish is common and widespread in Lao PDR.

Research Questions

How effective are the most salient species in repelling, and killing fly larvae in fermenting fish?

Material and Methods

The three plant species most frequently reported to repel fly larvae during an ethnobotanical survey throughout Lao PDR were tested for repellence and larvicidal activity of fly larvae infesting fermented fish. The lethality and repellence of Tadehagi triquetrum (L.) H. Ohashi (Fabaceae), Uraria crinita (L.) Desv. ex DC. (Fabaceae) and Bambusa multiplex (Lour.) Raeusch. ex Schult. & Schult. f. (Poaceae) were tested in an experimental design using fermenting fish in Vientiane, Lao PDR.

Results

The repellent effect of fresh material of T. triquetrum and U. crinita, and the larvicidal effect of fresh B. multiplex, is significantly more effective than that of dried material of the same species, and the total effect (repellence and larvicidal effect combined) for each of the three species was significantly more effective for fresh than for dry material. Fresh material of T. triquetrum, U. crinita, or B. multiplex added on top of the fermenting fish repelled 50%, 54%, 37%, and killed 22%, 28%, and 40% of fly larvae. The total effect was not significantly different per species at 72%, 82%, and 77%, respectively.

Discussion and Conclusions

The three most salient species are effective in repelling and killing fly larvae in the production of fermented fish, and may be essential to augment food safety during traditional fermentation in open jars.     

Evidence of a louse-borne outbreak involving typhus in Douai, 1710-1712 during the war of Spanish succession

Background

The new field of paleomicrobiology allows past outbreaks to be identified by testing dental pulp of human remains with PCR.

Methods

We identified a mass grave in Douai, France dating from the early XVIII^th century. This city was besieged during the European war of Spanish succession. We tested dental pulp from 1192 teeth (including 40 from Douai) by quantitative PCR (qPCR) for R. prowazekii and B. quintana. We also used ultra-sensitive suicide PCR to detect R. prowazekii and genotyped positive samples.

Results and Discussion

In the Douai remains, we identified one case of B. quintana infection (by qPCR) and R. prowazekii (by suicide PCR) in 6/21 individuals (29%). The R. prowazekii was genotype B, a genotype previously found in a Spanish isolate obtained in the first part of the XX^th century.

Conclusion

Louse-borne outbreaks were raging during the XVIII^th century; our results support the hypothesis that typhus was imported into Europe by Spanish soldiers from America.     

Atmospheric Hypoxia Limits Selection for Large Body Size in Insects

Background

The correlations between Phanerozoic atmospheric oxygen fluctuations and insect body size suggest that higher oxygen levels facilitate the evolution of larger size in insects.

Methods and Principal Findings

Testing this hypothesis we selected Drosophila melanogaster for large size in three oxygen atmospheric partial pressures (aPO₂). Fly body sizes increased by 15% during 11 generations of size selection in 21 and 40 kPa aPO₂. However, in 10 kPa aPO₂, sizes were strongly reduced. Beginning at the 12^th generation, flies were returned to normoxia. All flies had similar, enlarged sizes relative to the starting populations, demonstrating that selection for large size had functionally equivalent genetic effects on size that were independent of aPO₂.

Significance

Hypoxia provided a physical constraint on body size even in a tiny insect strongly selected for larger mass, supporting the hypothesis that Triassic hypoxia may have contributed to a reduction in insect size.     

Ganglioside GM3 Levels Are Altered in a Mouse Model of HIBM: GM3 as a Cellular Marker of the Disease

Objective

HIBM (Hereditary Inclusion Body Myopathy) is a recessive hereditary disease characterized by adult-onset, slowly progressive muscle weakness sparing the quadriceps. It is caused by a single missense mutation of each allele of the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) gene, a bifunctional enzyme catalyzing the first two steps of sialic acid synthesis in mammals. However, the mechanisms and cellular pathways affected by the GNE mutation and causing the muscle weakness could not be identified so far. Based on recent evidence in literature, we investigated a new hypothesis, i.e. the involvement in the disease of the GM3 ganglioside, a specific glycolipid implicated in muscle cell proliferation and differentiation.

Methods

qRT-PCR analysis of St3gal5 (GM3 synthase) gene expression and HPLC quantification of GM3 ganglioside were conducted on muscle tissue from a mouse model of HIBM harboring the M712T mutation of GNE (Gne^M712T/M712T mouse) vs control mice (Gne^+/+ mouse).

Results

St3gal5 mRNA levels were significantly lower in Gne^M712T/M712T mouse muscles vs Gne^+/+ mouse muscles (64.41%±10% of Gne^+/+ levels). GM3 ganglioside levels showed also a significant decrease in Gne^M712T/M712T mouse muscle compared to Gne^+/+ mouse muscle (18.09%±5.33% of Gne^+/+ levels). Although these Gne^M712T/M712T mice were described to suffer severe glomerular proteinuria, no GM3 alterations were noted in kidneys, highlighting a tissue specific alteration of gangliosides.

Conclusion

The M712T mutation of GNE hampers the muscle ability to synthesize normal levels of GM3. This is the first time that a mutation of GNE can be related to the molecular pathological mechanism of HIBM.     

Pulmonary Abnormalities in Mice with Paracoccidioidomycosis: A Sequential Study Comparing High Resolution Computed Tomography and Pathologic Findings

Background

Human paracoccidioidomycosis (PCM) is an endemic fungal disease of pulmonary origin. Follow-up of pulmonary lesions by image studies in an experimental model of PCM has not been previously attempted. This study focuses on defining patterns, topography and intensity of lung lesions in experimentally infected PCM mice by means of a comparative analysis between High Resolution Computed Tomography (HRCT) and histopathologic parameters.

Methodology

Male BALB/c mice were intranasally inoculated with 3×10⁶ Paracoccidioides brasiliensis (Pb) conidia (n = 50) or PBS (n = 50). HRCT was done every four weeks to determine pulmonary lesions, quantify lung density, reconstruct and quantify lung air structure. Lungs were also analyzed by histopathology and histomorphometry.

Results

Three different patterns of lesions were evidenced by HRCT and histopathology, as follows: nodular-diffuse, confluent and pseudo-tumoral. The lesions were mainly located around the hilus and affected more frequently the left lung. At the 4^th week post-challenge HRCT showed that 80% of the Pb-infected mice had peri-bronchial consolidations associated with a significant increase in upper lung density when compared with controls, (−263±25 vs. −422±10 HU, p<0.001). After the 8^th and 12^th weeks, consolidation had progressed involving also the middle regions. Histopathology revealed that consolidation as assessed by HRCT was equivalent histologically to a confluent granulomatous reaction, while nodules corresponded to individual compact granulomas. At the 16^th week of infection, confluent granulomas formed pseudotumoral masses that obstructed large bronchi. Discrete focal fibrosis was visible gradually around granulomas, but this finding was only evident by histopathology.

Conclusions/Significance

This study demonstrated that conventional HRCT is a useful tool for evaluation and quantification of pulmonary damage occurring in experimental mouse PCM. The experimental design used decreases the need to sacrifice a large number of animals, and serves to monitor treatment efficacy by means of a more rational approach to the study of human lung disease.     

High throughput, multiplexed pathogen detection authenticates plague waves in medieval Venice, Italy

Background

Historical records suggest that multiple burial sites from the 14th–16^th centuries in Venice, Italy, were used during the Black Death and subsequent plague epidemics.

Methodology/Principal Findings

High throughput, multiplexed real-time PCR detected DNA of seven highly transmissible pathogens in 173 dental pulp specimens collected from 46 graves. Bartonella quintana DNA was identified in five (2.9%) samples, including three from the 16th century and two from the 15th century, and Yersinia pestis DNA was detected in three (1.7%) samples, including two from the 14th century and one from the 16th century. Partial glpD gene sequencing indicated that the detected Y. pestis was the Orientalis biotype.

Conclusions

These data document for the first time successive plague epidemics in the medieval European city where quarantine was first instituted in the 14th century.     

Clinical Utility of a Commercial LAM-ELISA Assay for TB Diagnosis in HIV-Infected Patients Using Urine and Sputum Samples

Background

The accurate diagnosis of TB in HIV-infected patients, particularly with advanced immunosuppression, is difficult. Recent studies indicate that a lipoarabinomannan (LAM) assay (Clearview-TB®-ELISA) may have some utility for the diagnosis of TB in HIV-infected patients; however, the precise subgroup that may benefit from this technology requires clarification. The utility of LAM in sputum samples has, hitherto, not been evaluated.

Methods

LAM was measured in sputum and urine samples obtained from 500 consecutively recruited ambulant patients, with suspected TB, from 2 primary care clinics in South Africa. Culture positivity for M. tuberculosis was used as the reference standard for TB diagnosis.

Results

Of 440 evaluable patients 120/387 (31%) were HIV-infected. Urine-LAM positivity was associated with HIV positivity (p = 0.007) and test sensitivity, although low, was significantly higher in HIV-infected compared to uninfected patients (21% versus 6%; p<0.001), and also in HIV-infected participants with a CD4 <200 versus >200 cells/mm³ (37% versus 0%; p = 0.003). Urine-LAM remained highly specific in all 3 subgroups (95%–100%). 25% of smear-negative but culture-positive HIV-infected patients with a CD4 <200 cells/mm³ were positive for urine-LAM. Sputum-LAM had good sensitivity (86%) but poor specificity (15%) likely due to test cross-reactivity with several mouth-residing organisms including actinomycetes and nocardia species.

Conclusions

These preliminary data indicate that in a high burden primary care setting the diagnostic usefulness of urine-LAM is limited, as a rule-in test, to a specific patient subgroup i.e. smear-negative HIV-infected TB patients with a CD4 count <200 cells/mm³, who would otherwise have required further investigation. However, even in this group sensitivity was modest. Future and adequately powered studies in a primary care setting should now specifically target patients with suspected TB who have advanced HIV infection.     

Involvement of the Modifier Gene of a Human Mendelian Disorder in a Negative Selection Process

Background

Identification of modifier genes and characterization of their effects represent major challenges in human genetics. SAA1 is one of the few modifiers identified in humans: this gene influences the risk of renal amyloidosis (RA) in patients with familial Mediterranean fever (FMF), a Mendelian autoinflammatory disorder associated with mutations in MEFV. Indeed, the SAA1 α homozygous genotype and the p.Met694Val homozygous genotype at the MEFV locus are two main risk factors for RA.

Methodology/Principal Findings

Here, we investigated Armenian FMF patients and controls from two neighboring countries: Armenia, where RA is frequent (24%), and Karabakh, where RA is rare (2.5%). Sequencing of MEFV revealed similar frequencies of p.Met694Val homozygotes in the two groups of patients. However, a major deficit of SAA1 α homozygotes was found among Karabakhian patients (4%) as compared to Armenian patients (24%) (p = 5.10⁻⁵). Most importantly, we observed deviations from Hardy-Weinberg equilibrium (HWE) in the two groups of patients, and unexpectedly, in opposite directions, whereas, in the two control populations, genotype distributions at this locus were similar and complied with (HWE).

Conclusions/Significance

The excess of SAA1α homozygotes among Armenian patients could be explained by the recruitment of patients with severe phenotypes. In contrast, a population-based study revealed that the deficit of α/α among Karabakhian patients would result from a negative selection against carriers of this genotype. This study, which provides new insights into the role of SAA1 in the pathophysiology of FMF, represents the first example of deviations from HWE and selection involving the modifier gene of a Mendelian disorder.     

Silencing of aphid genes by dsRNA feeding from plants

Background

RNA interference (RNAi) is a valuable reverse genetics tool to study gene function in various organisms, including hemipteran insects such as aphids. Previous work has shown that RNAi-mediated knockdown of pea aphid (Acyrthosiphon pisum) genes can be achieved through direct injection of double-stranded RNA (dsRNA) or small-interfering RNAs (siRNA) into the pea aphid hemolymph or by feeding these insects on artificial diets containing the small RNAs.

Methodology/Principal Findings

In this study, we have developed the plant-mediated RNAi technology for aphids to allow for gene silencing in the aphid natural environment and minimize handling of these insects during experiments. The green peach aphid M. persicae was selected because it has a broad plant host range that includes the model plants Nicotiana benthamiana and Arabidopsis thaliana for which transgenic materials can relatively quickly be generated. We targeted M. persicae Rack1, which is predominantly expressed in the gut, and M. persicae C002 (MpC002), which is predominantly expressed in the salivary glands. The aphids were fed on N. benthamiana leaf disks transiently producing dsRNA corresponding to these genes and on A. thaliana plants stably producing the dsRNAs. MpC002 and Rack-1 expression were knocked down by up to 60% on transgenic N. benthamiana and A. thaliana. Moreover, silenced M. persicae produced less progeny consistent with these genes having essential functions.

Conclusions/Significance

Similar levels of gene silencing were achieved in our plant-mediated RNAi approach and published silencing methods for aphids. Furthermore, the N. benthamiana leaf disk assay can be developed into a screen to assess which genes are essential for aphid survival on plants. Our results also demonstrate the feasibility of the plant-mediated RNAi approach for aphid control.