Male Midguts Research Articles

Characterization of midgut microbiome of Anopheles stephensi Liston.

Anopheles stephensi is an important vector of malaria in South East Asia. The abundance and diversity of gut microbiota in the disease vectors affect their development, digestion, metabolism and immunity. The immatures of An. stephensi engulf microbes from their aquatic environment. The present study investigates midgut microbiota of wild and laboratory populations and compares it with their habitat bacterial diversity to study transstadial transmissibility. The gut microbes from immatures, adults and water samples were cultured at ambient conditions on different media. The colony and biochemical characteristics, and 16S rRNA gene sequencing of gut microbes were studied. Altogether, 298 bacterial isolates were characterized as 21 genera belonging to four major Phyla viz., Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria. In the field population-1, Proteobacteria and Firmicutes accounted for 49% and Actinobacteria constituted 51% of the bacterial isolates. In field population-2, Bacteroidetes and Firmicutes accounted for 99% of the isolates. In the laboratory populations, Firmicutes constituted 77%, while Proteobacteria 23% of the isolates. Additionally, 9 genera occurred in the breeding habitats, 13 in the larval midgut, 6 in pupal midgut, 9 in male midgut and 10 in the female midgut. This is a unique study on diversity of microbiota of An. stephensi from breeding water, developmental stages and adults. Different culture media used enhanced the isolation of diverse bacteria. The presence of Micrococcus and Leucobacter in different life stages indicates their adaptation in An. stephensi as symbionts which need further evaluation for their role in paratransgenesis.

Insights into the Gryllus bimaculatus Immune-Related Transcriptomic Profiling to Combat Naturally Invading Pathogens.

Natural pathogen pressure is an important factor that shapes the host immune defense mechanism. The current study primarily aimed to explore the molecular basis of the natural immune defense mechanism of a sporadic pest, Gryllus bimaculatus, during swarming by constructing cDNA libraries of the female mid-gut, male mid-gut, testes, and ovaries. The Illumina HiSeq platform generated an average of 7.9 G, 11.77 G, 10.07 G, and 10.07 G bases of outputs from the male mid-gut, female mid-gut, testes, and ovaries and libraries, respectively. The transcriptome of two-spotted field crickets was assembled into 233,172 UniGenes, which yielded approximately 163.58 million reads. On the other hand, there were 43,055 genes in common that were shared among all the biological samples. Gene Ontology analysis successfully annotated 492 immune-related genes, which comprised mainly Pattern Recognition Receptors (62 genes), Signal modulators (57 genes), Signal transduction (214 genes), Effectors (36 genes), and another immune-related 123 genes. In summary, the identified wide range of immune-related genes from G. bimaculatus indicates the existence of a sophisticated and specialized broad spectrum immune mechanism against invading pathogens, which provides, for the first time, insights into the molecular mechanism of disease resistance among two-spotted field crickets.

First study on lipid dynamics during the female reproductive cycle ofPolybetes pythagoricus(Araneae: Sparassidae)

Spiders are valuable to humans, not only for their role in health but also as biologic pest controllers. In oviparous species, lipids are the main energy source for embryo development and the growth and survival of larvae. Using the spider Polybetes pythagoricus (Holmberg, 1875) as an experimental model, we studied the fluctuations in lipids and fatty acids occurring in tissues related to vitellogenesis. Different reproductive stages (previtellogenesis, early vitellogenesis, vitellogenesis, and postvitellogenesis) were determined histologically. Gonadosomatic and hepatosomatic indices were first used in spiders. The midgut diverticula proved to be the organ with the highest lipid concentration, with triacylglycerols as the major component. Phospholipids were the principal lipids transported. In vitellogenesis, a major accumulation of lipids occurred in the ovary, principally phosphatidylethanolamine (41%); it probably synthesized in the midgut diverticula before being released into the hemolymph for transport and accumulation in the ovary. Phosphatidylethanolamine is possibly involved in maintaining membrane fluidity and in the function of the electron transport chain. The principal fatty acids in the different organs were palmitic, stearic, oleic, and linoleic acids. During vitellogenesis, the ovaries become enriched in polyunsaturated fatty acids. The lipid patterns in the male midgut diverticula, muscle, and hemolymph were similar to those of the previtellogenic or postvitellogenic females.

PhaR, a Negative Regulator of PhaP, Modulates the Colonization of a Burkholderia Gut Symbiont in the Midgut of the Host Insect, Riptortus pedestris.

Five genes encoding PhaP family proteins and one phaR gene have been identified in the genome of Burkholderia symbiont strain RPE75. PhaP proteins function as the surface proteins of polyhydroxyalkanoate (PHA) granules, and the PhaR protein acts as a negative regulator of PhaP biosynthesis. Recently, we characterized one phaP gene to understand the molecular cross talk between Riptortus insects and Burkholderia gut symbionts. In this study, we constructed four other phaP gene-depleted mutants (ΔphaP1, ΔphaP2, ΔphaP3, and ΔphaP4 mutants), one phaR gene-depleted mutant, and a phaR-complemented mutant (ΔphaR/phaR mutant). To address the biological roles of four phaP family genes and the phaR gene during insect-gut symbiont interaction, these Burkholderia mutants were fed to the second-instar nymphs, and colonization ability and fitness parameters were examined. In vitro, the ΔphaP3 and ΔphaR mutants cannot make a PHA granule normally in a stressful environment. Furthermore, the ΔphaR mutation decreased the colonization ability in the host midgut and negatively affected the host insect's fitness compared with wild-type Burkholderia-infected insects. However, other phaP family gene-depleted mutants colonized well in the midgut of the fifth-instar nymph insects. However, in the case of females, the colonization rate of the ΔphaP3 mutant was decreased and the host's fitness parameters were decreased compared with the wild-type-infected host, suggesting that the environment of the female midgut may be more hostile than that of the male midgut. These results demonstrate that PhaR plays an important role in the biosynthesis of PHA granules and that it is significantly related to the colonization of the Burkholderia gut symbiont in the host insects' midgut.IMPORTANCE Bacterial polyhydroxyalkanoate (PHA) biosynthesis is a complex process requiring several enzymes. The biological roles of PHA granule synthesis enzymes and the surface proteins of PHA granules during host-gut symbiont interactions are not fully understood. Here, we report the effects on colonization ability in the host midguts and the fitness of host insects after feeding Burkholderia mutant cells (four phaP-depleted mutants and one phaR-depleted mutant) to the host insects. Analyses of both synthesized PHA granule amounts and CFU numbers suggest that the phaR gene is closely related to synthesis of the PHA granule and the colonization of the Burkholderia gut symbiont in the host insect's midgut. Like our previous report, this study also supports the idea that the environment of the host midgut may not be favorable to symbiotic Burkholderia cells and that PHA granules may be required to adapt in the host midgut.

Steps in the Biosynthesis of Fuscumol in the Longhorn Beetles Tetropium fuscum (F.) and Tetropium cinnamopterum Kirby

Fuscumol [(2S,5E)-6,10-dimethyl-5,9-undecadien-2-ol] was recently identified as the male-produced aggregation pheromone of the brown spruce longhorn beetle, Tetropium fuscum (F.), and the eastern larch borer, Tetropium cinnamopterum Kirby. Several other species use this homoterpenoid alcohol motif, its ketone, or its acetate as part of their pheromone system. Investigation of the biosynthesis of this compound in these two Tetropium species demonstrated that geranylacetone [(5E)-6,10-dimethyl-5,9-undecadien-2-one] and farnesol [(2E,6E)-3,7,11-trimethyl-2,6,10-dodecatrien-1-ol] are both intermediates in this process. This was accomplished by applying deuterium-labeled geranylacetone and deuterium-labeled farnesol in separate experiments to the abdominal sterna of live T. fuscum and T. cinnamopterum and analyzing the deuterium labeling in the fuscumol and geranylacetone emitted by the insects with solid-phase microextraction (SPME) and GC/MS analysis. Deuterium labeling studies also showed that nerolidol[(3S,6E)-3-hydroxy-3,7,11-trimethyl-1,6,10-dodecatriene] and 2,3-epoxyfarnesol are not intermediates in fuscumol or geranylacetone synthesis in T. fuscum or T. cinnamopterum. Tissue-specific expression of T. fuscum farnesyl diphosphate synthase (TfFPPS), an enzyme expected to provide a key fuscumol precursor, was measured. TfFPPS transcripts were relatively abundant in male midguts, but were also present at significant levels in other tissues.

Ipsdienol dehydrogenase (IDOLDH): A novel oxidoreductase important for Ips pini pheromone production

Ipsdienone (2-methyl-6-methylene-2,7-octadien-4-one) is an important intermediate in the biosynthesis of pheromonal ipsdienol (2-methyl-6-methylene-2,7-octadien-4-ol) and ipsenol (2-methyl-6-methylene-7-octen-4-ol) in male pine engraver beetles, Ips pini (Say). A novel ipsdienol dehydrogenase (IDOLDH) with a pheromone-biosynthetic gene expression pattern was cloned, expressed, functionally characterized, and its cellular localization analyzed. The cDNA has a 762 nt ORF encoding a 253 amino acid predicted translation product of 28 kDa and pI 5.8. The protein has conserved motifs of the Cp2 subfamily of “classical” short-chain dehydrogenases. Transcript levels were highest in pheromone producing tissue: the anterior midgut of fed males. The protein was detected only in male midguts and localized in the cytosolic fraction of midgut cells. Recombinant IDOLDH was produced in Sf9 cells using a baculovirus expression system. Enzyme assays of protein preparations showed IDOLDH used both NAD⁺ and NADP⁺ as coenzymes with specific activities in the nanomole range. Enzyme assays and GC/MS analysis showed that IDOLDH catalyzed the oxidation of racemic ipsdienol and (4 R)-(−)-ipsdienol to form ipsdienone, while (4 S)-(+)-ipsdienol was not a substrate. These data strongly implicate IDOLDH as an enzyme involved in terminal pheromone-biosynthetic steps, likely functioning to “tune” ipsdienol enantiomeric ratios.

Isolation, endocrine regulation and transcript distribution of a putative primary JH-responsive gene from the pine engraver, Ips pini (Coleoptera: Scolytidae)

We isolated a cDNA of unknown function from a juvenile hormone III (JH III)-treated male midgut cDNA library prepared from the pine engraver beetle, Ips pini, and examined its genomic structure. The gene, tentatively named “ Ipi10G08”, encoded a 410 amino acid translation product that shared 26–37% identity with unannotated matches from several insects. Semi-quantitative RT-PCR analysis of Ipi10G08 following application of a 10 μg dose of JH III demonstrated an early induction for both male and female beetles, with transcripts being detectable after 45 min. An expression profile of male midgut tissue indicated Ipi10G08 transcript levels reach a maximum induction of ∼22.5-fold control levels at 4 h post-treatment. Tissue distribution studies displayed a large induction of Ipi10G08 mRNA in the alimentary canal of JH III-treated beetles, especially in males. A dose curve from both sexes suggested there may be a difference in the ability to respond to lower levels of JH III and immunoblot analysis indicated that although JH III highly induces transcript levels in females, protein levels are not similarly induced, while protein levels are induced in males. Ipi10G08 is likely a primary JH response gene and may provide insight into how this hormone exerts its actions.

Spatial and sex-specific dissection of the Anopheles gambiae midgut transcriptome

BackgroundThe midgut of hematophagous insects, such as disease transmitting mosquitoes, carries out a variety of essential functions that mostly relate to blood feeding. The midgut of the female malaria vector mosquito Anopheles gambiae is a major site of interactions between the parasite and the vector. Distinct compartments and cell types of the midgut tissue carry out specific functions and vector borne pathogens interact and infect different parts of the midgut.ResultsA microarray based global gene expression approach was used to compare transcript abundance in the four major female midgut compartments (cardia, anterior, anterior part of posterior and posterior part of posterior midgut) and between the male and female Anopheles gambiae midgut. Major differences between the female and male midgut gene expression relate to digestive processes and immunity. Each compartment has a distinct gene function profile with the posterior midgut expressing digestive enzyme genes and the cardia and anterior midgut expressing high levels of antimicrobial peptide and other immune gene transcripts. Interestingly, the cardia expressed several known anti-Plasmodium factors. A parallel peptidomic analysis of the cardia identified known mosquito antimicrobial peptides as well as several putative short secreted peptides that are likely to represent novel antimicrobial factors.ConclusionThe A. gambiae sex specific midgut and female midgut compartment specific transcriptomes correlates with their known functions. The significantly greater functional diversity of the female midgut relate to hematophagy that is associated with digestion and nutrition uptake as well as exposes it to a variety of pathogens, and promotes growth of its endogenous microbial flora. The strikingly high proportion of immunity related factors in the cardia tissue most likely serves the function to increase sterility of ingested sugar and blood. A detailed characterization of the functional specificities of the female mosquito midgut and its various compartments can greatly contribute to our understanding of its role in disease transmission and generate the necessary tools for the development of malaria control strategies.

Functional expression of a bark beetle cytochrome P450 that hydroxylates myrcene to ipsdienol

The final steps in the pheromone-biosynthetic pathway of the pine engraver beetle, Ips pini (Say) (Coleoptera: Scolytidae) are unknown, but likely involve myrcene (7-methyl-3-methylene-1,6-octadiene) hydroxylation to produce the aggregation pheromone component, ipsdienol (2-methyl-6-methylene-2,7-octadien-4-ol). We have isolated a full-length I. pini cDNA encoding a cytochrome P450, CYP9T2. The recovered cDNA is 1.83 kb and the open reading frame encodes a 532 amino acid protein. CYP9T2 is regulated by the same physiological factors that induce pheromone production. Quantitative real-time PCR experiments showed that feeding on host phloem induced CYP9T2 expression in males, but not females, and that basal expression levels are highest in male midguts, similar to other I. pini pheromone-biosynthetic genes. Microsomes prepared from Sf9 cells co-expressing baculoviral-mediated recombinant CYP9T2 and housefly ( Musca domestica) NADPH-cytochrome P450 reductase converted myrcene to ipsdienol. The product identified by coupled GC-MS was mostly (4 R)-(−)-ipsdienol, an important aggregation pheromone component for western North American I. pini. These results are consistent with CYP9T2 encoding a myrcene hydroxylase that functions near the end of the pheromone-biosynthetic pathway.

Coordinated gene expression for pheromone biosynthesis in the pine engraver beetle, Ips pini (Coleoptera: Scolytidae).

In several pine bark beetle species, phloem feeding induces aggregation pheromone production to coordinate a mass attack on the host tree. Male pine engraver beetles, Ips pini (Say) (Coleoptera: Scolytidae), produce the monoterpenoid pheromone component ipsdienol de novo via the mevalonate pathway in the anterior midgut upon feeding. To understand how pheromone production is regulated in this tissue, we used quantitative real-time PCR to examine feeding-induced changes in gene expression of seven mevalonate pathway genes: acetoacetyl-coenzyme A thiolase, 3-hydroxy-3-methylglutaryl coenzyme A synthase, 3-hydroxy-3-methylglutaryl coenzyme A reductase, mevalonate 5-diphosphate decarboxylase, isopentenyl-diphosphate isomerase, geranyl-diphosphate synthase (GPPS), and farnesyl-diphosphate synthase (FPPS). In males, expression of all these genes significantly increased upon feeding. In females, the expression of the early mevalonate pathway genes (up to and including the isomerase) increased significantly, but the expression of the later genes (GPPS and FPPS) was unaffected or decreased upon feeding. Thus, feeding coordinately regulates expression of the mevalonate pathway genes necessary for pheromone biosynthesis in male, but not female, midguts. Furthermore, basal mRNA levels were 5- to 41-fold more abundant in male midguts compared to female midguts. This is the first report of coordinated regulation of mevalonate pathway genes in an invertebrate model consistent with their sex-specific role in de novo pheromone biosynthesis.

The distribution and myotropic activity of locustatachykinin-like peptides in locust midgut

The midgut of the African migratory locust, Locusta migratoria, was found to contain endocrine-like cells that stained positively for locustatachykinin I (Lom TK I)-like immunoreactivity. These cells were distributed in an unequal manner throughout the midgut of the locust, with a greater density of Lom TK I-like immunoreactive endocrine-like cells occurring in the posterior region of the midgut. These singly occurring cells appear elongate with an apical extension projecting toward the midgut lumen and a smaller projection extending towards the midgut basal lamina. No immunoreactive neuronal processes were detected along the midgut wall. Radioimmunoassays revealed that the female midgut contained two to three times more Lom TK I-like material than the male midgut, and radioimmunoassay coupled to high-performance liquid chromatography analysis revealed that at least five locustatachykinin isoforms appear to be present in the midgut. This distribution of Lom TK I-like material suggests possible functional differences in the various regions of the midgut. The role that these cells may play in locust midgut secretory activity and motility remains unknown. However, the addition of synthetic Lom TK I through IV to a ring type midgut muscle preparation stimulated contraction of midgut circular muscles, suggesting a possible physiological role for these peptides. Dose-response curves constructed for Lom TK I-IV revealed that the peptide-induced contractions increased in a dose-dependent manner.

Analysis of stage‐specific and shared antigens derived from Rhipicephalus sanguineus by electrophoresis and Western blotting

We carried out an SDS-PAGE analysis of antigens of Rhipicephalus sanguineus using extracts of eggs (EE), larvae (LE), nymphs (NE), male salivary glands (MSGE), male midguts (MME), female salivary glands (FSGE) and female midguts (FME). Under non-reducing conditions a common band of about 205 kDa was observed. EE, LE and NE extracts showed groups of bands between 150 and 75 kDa. A protein pattern was observed in FSGE extract with a group of bands between 75 and 50 kDa and four bands between 15 and 6.5 kDa. In this case an apparently exclusive band of molecular weight about 25 kDa was observed. Under reducing conditions similarities between LE and NE extracts increased, separating from the EE pattern. On the other hand, we have determined the presence of stage-specific and common antigens on EE, LE, NE, MSGE, MME, FSGE and FME extracts of R.sanguineus by means of immunoblots using polyclonal sera of rabbits infested with larvae, nymphs or adults of this tick. EE extract was only recognized by the anti-larva sera. Higher reactivity was observed when the extracts were tested with anti-adult sera. In these experiments a very prominent band of molecular weight about 45 kDa was detected. This band was not observed under reducing conditions. Higher reactivity with anti-adult sera was observed against FSGE extract.

Immunological response in rabbits infested with Rhipicephalus sanguineus

In order to clarify the importance of humoral antibody in host resistance to ticks, in the present work we studied the immunological response of rabbits infested with larvae, nymphs or adults of Rhipicephalus sanguineus, using extracts of eggs (EE), larvae (LE), nymphs (NE), male salivary glands (MSGE), male midguts (MME), female salivary glands (FSGE) and female midguts (FME). When serum from rabbits infested with larvae or nymphs was tested using enzyme-linked immunosorbent assay, no reactions were observed with any of the extracts including the homologous LE or NE. In sera from rabbits infested with adult ticks, the reactions were observed in both homologous (MSGE, MME, FSGE and FME) and heterologous (EE, LE and NE) system. However, differences were seen regarding the type of antigen used. When the experiment was carried out using extracts from adults higher responses were found. With FSGE and FME antigens, antibody levels were systematically higher than those observed when MSGE and MME were used.