Palpigrades from Cuba (Arachnida: Palpigradi: Eukoeneniidae).

The knowledge of palpigrades in Cuba is limited to the species Eukoenenia orghidani, discovered and described from Cueva de Bellamar. In this work, a survey for palpigrades in the suburbs of La Habana, Cuba revealed three species, Eukoenenia berlesei, Eukoenenia florenciae and a new species described here as Eukoenenia glandulosa sp. nov. Interestingly, the three species coexist in the microspaces of wet soil. A total of 16 arthropod species were identified living in the same microhabitat as the palpigrades.

New and interesting palpigrades (Arachnida, Palpigradi) of the genera Koeneniodes Silvestri, 1913 and Prokoenenia Börner, 1901 from Asia.

Two new species of palpigrades are described: a soil-dwelling species of the genus Koeneniodes Silvestri, 1913 from a broadleaf forest in Tibet and an extraordinary cave-dwelling species from Jinhua cave in China belonging to Prokoenenia Börner, 1901. Koeneniodes tibetanus sp. n. is related to Koeneniodes spiniger from Thailand. The two species share the presence of four thick and spiniform setae on the second lobe of the female genitalia; they differ in the number of thick setae on opisthosomal sternite IV, the number of cheliceral teeth, the coxal setal formula, and the morphology of the spiniform setae. Prokoenenia sarcodactylica sp. n. is based on an immature female from Jinhua Cave, Beijing. The presence of 18 finger-shaped blades in the lateral organs-unique among palpigrades , the large body size (2150 µm) and the extremely long basitarsus IV (205 µm) indicate that the new species is the first undoubtedly caveadapted Prokoenenia. This is also the first record of the genus Prokoenenia from China.

A new troglobitic palpigrade from Central Brazil, with notes on a new opisthosomal character (Arachnida: Palpigradi).

A new species of palpigrade from the cave Gruta Cabeceira d'água in Goiás state in Central Brazil is described and illustrated based on 11 specimens: five adult males, two adult females, two immatures C and two immatures B. Eukoenenia audax sp. nov. was collected mainly in the sand banks near the main water stream, where flooding and important water fluctuations occur in the cave. The new Brazilian species shows three uncommon morphological traits: the presence of 7 + 7 setae on the propeltidium, one deuto-tritosternal seta, and 9-11 blades in the lateral organs; these characters relate Eukoenenia audax sp. nov. with only a handful of species in the world. The presence of a pair of cavities was identified in the intersegmental furrow between opisthosomal sternites III-IV, IV-V, V-VI and VI-VII. A comprehensive study of this new character was performed in other species available to the authors, and a discussion of its possible origin and function is presented.

A review of larval Chyzeria Canestrini, 1897 (Acari: Parasitengonina: Chyzeriidae).

Type-material for all larvae of species of Chyzeria Canestrini, 1897 (Prostigmata: Chyzerioidea: Chyzeriidae) was examined, diagnoses are presented and a key to species provided. The genus Chyzeria is divided in two species groups, onychia and hirsti, according to morphological and host preference differences. In addition, two new species of Chyzeria are described from specimens parasitising paropsine beetles (Coleoptera: Chrysomelidae): Chyzeria southcotti n. sp. from five species of Paropsini in northern New South Wales, and Chyzeria grandis n. sp. from Paropsisterna agricola (Chapuis) in Victoria and Trachymela sp. in Tasmania. The larvae of Chyzeria derricki Southcott, 1982 are reported from Anostostoma australasiae (Gray) (Orthoptera: Stenopelmatidae) in southeast Queensland, and larvae of Chyzeria flindersi Southcott, 1982 are reported from P. agricola from Tasmania.

Wolbachia small noncoding RNAs and their role in cross-kingdom communications.

In prokaryotes, small noncoding RNAs (snRNAs) of 50-500 nt are produced that are important in bacterial virulence and response to environmental stimuli. Here, we identified and characterized snRNAs from the endosymbiotic bacteria, Wolbachia, which are widespread in invertebrates and cause reproductive manipulations. Most importantly, some strains of Wolbachia inhibit replication of several vector-borne pathogens in insects. We demonstrate that two abundant snRNAs, WsnRNA-46 and WsnRNA-49, are expressed in Wolbachia from noncoding RNA transcripts that contain precursors with stem-loop structures. WsnRNAs were detected in Aedes aegypti mosquitoes infected with the wMelPop-CLA strain of Wolbachia and in Drosophila melanogaster and Drosophila simulans infected with wMelPop and wAu strains, respectively, indicating that the WsnRNAs are conserved across species and strains. In addition, we show that the WsnRNAs may potentially regulate host genes and Wolbachia genes. Our findings provide evidence for the production of functional snRNAs by Wolbachia that play roles in cross-kingdom communication between the endosymbiont and the host.

Wolbachia infection modifies the profile, shuttling and structure of microRNAs in a mosquito cell line.

MicroRNAs (miRNAs) are small non-coding RNAs that play important roles in many biological processes such as development, cell signaling and immune response. Small RNA deep sequencing technology provided an opportunity for a thorough survey of the miRNA profile of a mosquito cell line from Aedes aegypti. We characterized the miRNA composition of the nucleus and the cytoplasm of uninfected cells and compared it with the one of cells infected with the endosymbiotic bacterium Wolbachia strain wMelPop-CLA. We found an overall increase of small RNAs between 18 and 28 nucleotides in both cellular compartments in Wolbachia-infected cells and identified specific miRNAs induced and/or suppressed by the Wolbachia infection. We discuss the mechanisms that the cell may use to shuttle miRNAs between the cytoplasm and the nucleus. In addition, we identified piRNAs that changed their abundance in response to Wolbachia infection. The miRNAs and piRNAs identified in this study provide promising leads for investigations into the host-endosymbiont interactions and for better understanding of how Wolbachia manipulates the host miRNA machinery in order to facilitate its persistent replication in infected cells.

Farnesyl phosphatase, a Corpora allata enzyme involved in juvenile hormone biosynthesis in Aedes aegypti.

BACKGROUND: The juvenile hormones (JHs) are sesquiterpenoid compounds that play a central role in insect reproduction, development and behavior. The late steps of JH III biosynthesis in the mosquito Aedes aegypti involve the hydrolysis of farnesyl pyrophosphate (FPP) to farnesol (FOL), which is then successively oxidized to farnesal and farnesoic acid, methylated to form methyl farnesoate and finally transformed to JH III by a P450 epoxidase. The only recognized FPP phosphatase (FPPase) expressed in the corpora allata (CA) of an insect was recently described in Drosophila melanogaster (DmFPPase). In the present study we sought to molecularly and biochemically characterize the FPP phosphatase responsible for the transformation of FPP into FOL in the CA of A. aegypti. METHODS: A search for orthologs of the DmFPPase in Aedes aegypti led to the identification of 3 putative FPPase paralogs expressed in the CA of the mosquito (AaFPPases-1, -2, and -3). The activities of recombinant AaFPPases were tested against general phosphatase substrates and isoprenoid pyrophosphates. Using a newly developed assay utilizing fluorescent tags, we analyzed AaFPPase activities in CA of sugar and blood-fed females. Double-stranded RNA (dsRNA) was used to evaluate the effect of reduction of AaFPPase mRNAs on JH biosynthesis. CONCLUSIONS: AaFPPase-1 and AaFPPase-2 are members of the NagD family of the Class IIA C2 cap-containing haloalkanoic acid dehalogenase (HAD) super family and efficiently hydrolyzed FPP into FOL. AaFPPase activities were different in CA of sugar and blood-fed females. Injection of dsRNAs resulted in a significant reduction of AaFPPase-1 and AaFPPase-2 mRNAs, but only reduction of AaFPPase-1 caused a significant decrease of JH biosynthesis. These results suggest that AaFPPase-1 is predominantly involved in the catalysis of FPP into FOL in the CA of A. aegypti.

Functional analysis of a mosquito short-chain dehydrogenase cluster.

The short-chain dehydrogenases (SDR) constitute one of the oldest and largest families of enzymes with over 46,000 members in sequence databases. About 25% of all known dehydrogenases belong to the SDR family. SDR enzymes have critical roles in lipid, amino acid, carbohydrate, hormone, and xenobiotic metabolism as well as in redox sensor mechanisms. This family is present in archaea, bacteria, and eukaryota, emphasizing their versatility and fundamental importance for metabolic processes. We identified a cluster of eight SDRs in the mosquito Aedes aegypti (AaSDRs). Members of the cluster differ in tissue specificity and developmental expression. Heterologous expression produced recombinant proteins that had diverse substrate specificities, but distinct from the conventional insect alcohol (ethanol) dehydrogenases. They are all NADP⁺-dependent and they have S-enantioselectivity and preference for secondary alcohols with 8-15 carbons. Homology modeling was used to build the structure of AaSDR1 and two additional cluster members. The computational study helped explain the selectivity toward the (10S)-isomers as well as the reduced activity of AaSDR4 and AaSDR9 for longer isoprenoid substrates. Similar clusters of SDRs are present in other species of insects, suggesting similar selection mechanisms causing duplication and diversification of this family of enzymes.

A new genus and species of larval mite (Acari: Prostigmata: Microtrombidiidae) parasitising Orthoptera (Tettigoniidae) from the Sierra Nevada, Spain.

Nevada capileirarum n. g., n. sp. (Acari: Microtrombidiidae: Microtrombidiinae) is described from ectoparasitic larvae parasitising two endemic species of Orthoptera (Tettigoniidae), Baetica ustulata (Rambur) and Pycnogaster inermis (Rambur) from the Sierra Nevada mountain range, Granada, Spain. A key to the larvae of microtrombidiine genera with three dorsal scuta and a coxal setal formula of 2-1-1 is presented.

Characterization of an isopentenyl diphosphate isomerase involved in the juvenile hormone pathway in Aedes aegypti.

Isopentenyl diphosphate isomerase (IPPI) is an enzyme involved in the synthesis of juvenile hormone (JH) in the corpora allata (CA) of insects. IPPI catalyzes the conversion of isopentenyl pyrophosphate (IPP) to dimethylallyl pyrophosphate (DMAPP); afterward IPP and DMAPP condense in a head-to-tail manner to produce geranyl diphosphate (GPP), this head-to-tail condensation can be repeated, by the further reaction of GPP with IPP, yielding the JH precursor farnesyl diphosphate. An IPPI expressed sequence tag (EST) was obtained from an Aedes aegypti corpora-allata + corpora cardiaca library. Its full-length cDNA encodes a 244-aa protein that shows a high degree of similarity with type I IPPIs from other organisms, particularly for those residues that have important roles in catalysis, metal coordination and interaction with the diphosphate moiety of the IPP. Heterologous expression produced a recombinant protein that metabolized IPP into DMAPP; treatment of DMAPP with phosphoric acid produced isoprene, a volatile compound that was measured with an assay based on a solid-phase micro extraction protocol and direct analysis by gas chromatography. A. aegypti IPPI (AaIPPI) required Mg(2+) or Mn(2+) but not Zn(2+) for full activity and it was entirely inhibited by iodoacetamide. Real time PCR experiments showed that AaIPPI is highly expressed in the CA. Changes in AaIPPI mRNA levels in the CA in the pupal and adult female mosquito corresponded well with changes in JH synthesis (Li et al., 2003). This is the first molecular and functional characterization of an isopentenyl diphosphate isomerase involved in the production of juvenile hormone in the CA of an insect.

Functional characterization of an allatotropin receptor expressed in the corpora allata of mosquitoes.

Allatotropin is an insect neuropeptide with pleiotropic actions on a variety of different tissues. In the present work we describe the identification, cloning and functional and molecular characterization of an Aedes aegypti allatotropin receptor (AeATr) and provide a detailed quantitative study of the expression of the AeATr gene in the adult mosquito. Analysis of the tissue distribution of AeATr mRNA in adult female revealed high transcript levels in the nervous system (brain, abdominal, thoracic and ventral ganglia), corpora allata-corpora cardiaca complex and ovary. The receptor is also expressed in heart, hindgut and male testis and accessory glands. Separation of the corpora allata (CA) and corpora cardiaca followed by analysis of gene expression in the isolated glands revealed expression of the AeATr primarily in the CA. In the female CA, the AeATr mRNA levels were low in the early pupae, started increasing 6h before adult eclosion and reached a maximum 24h after female emergence. Blood feeding resulted in a decrease in transcript levels. The pattern of changes of AeATr mRNA resembles the changes in JH biosynthesis. Fluorometric Imaging Plate Reader recordings of calcium transients in HEK293 cells expressing the AeATr showed a selective response to A. aegypti allatotropin stimulation in the low nanomolar concentration range. Our studies suggest that the AeATr play a role in the regulation of JH synthesis in mosquitoes.

A coordinated expression of biosynthetic enzymes controls the flux of juvenile hormone precursors in the corpora allata of mosquitoes.

Juvenile hormone (JH) is a key regulator of metamorphosis and ovarian development in mosquitoes. Adult female Aedes aegypti mosquitoes show developmental and dynamically regulated changes of JH synthesis. Newly emerged females have corpora allata (CA) with low biosynthetic activity, but they produce high amounts of JH a day later; blood feeding results in a striking decrease in JH synthesis, but the CA returns to a high level of JH synthesis three days later. To understand the molecular bases of these dynamic changes we combined transcriptional studies of 11 of the 13 enzymes of the JH pathway with a functional analysis of JH synthesis. We detected up to a 1000-fold difference in the levels of mRNA in the CA among the JH biosynthetic enzymes studied. There was a coordinated expression of the 11 JH biosynthetic enzymes in female pupae and adult mosquito. Increases or decreases in transcript levels for all the enzymes resulted in increases or decreases of JH synthesis; suggesting that transcript changes are at least partially responsible for the dynamic changes of JH biosynthesis observed. JH synthesis by the CA was progressively increased in vitro by addition of exogenous precursors such as geranyl-diphosphate, farnesyl-diphosphate, farnesol, farnesal and farnesoic acid. These results suggest that the supply of these precursors and not the activity of the last 6 pathway enzymes is rate limiting in these glands. Nutrient reserves play a key role in the regulation of JH synthesis. Nutritionally deficient females had reduced transcript levels for the genes encoding JH biosynthetic enzymes and reduced JH synthesis. Our studies suggest that JH synthesis is controlled by the rate of flux of isoprenoids, which is the outcome of a complex interplay of changes in precursor pools, enzyme levels and external regulators such as nutrients and brain factors. Enzyme levels might need to surpass a minimum threshold to achieve a net flux of precursors through the biosynthetic pathway. In glands with low synthetic activity, the flux of isoprenoids might be limited by the activity of enzymes with low levels of expression.

Inferior mesenteric artery aneurysm in the setting of chronic colonic vascular ectasia.

Colonic vascular ectasia is a condition characterized by dilated submucosal veins, venules, or capillaries found commonly in patients with lower gastrointestinal hemorrhage. We present a case of colorectal ectasia associated with ischemia and an inferior mesenteric artery aneurysm. These pathologic findings may be the result of the vascular ectasia and may add to the natural history of this condition.

Rapid direct analysis in real time (DART) mass spectrometric detection of juvenile hormone III and its terpene precursors.

Direct analysis in real time (DART) is a plasma-based ambient ionization technique that enables rapid ionization of small molecules with high sample throughput. In this work, DART was coupled to an orthogonal (oa) time-of-flight (TOF) mass spectrometer and the system was optimized for analyzing a vital hormonal regulator in insects, juvenile hormone (JH) III and its terpene precursors, namely, farnesol, farnesoic acid, and methyl farnesoate. Optimization experiments were planned using design of experiments (DOE) full factorial models to identify the most significant DART variables contributing to JH III analysis sensitivity by DART-TOF mass spectrometry (MS). The optimized DART-TOF MS method had femtomole to sub-picomole detection limits for terpene standards, along with mass accuracies below 5 ppm. Finally, the possibility of distinguishing between two farnesol isomers by in-source-collision-induced dissociation (CID) in the first differentially pumped region of the oaTOF mass spectrometer was investigated. DART-MS enabled high-throughput, sensitive analysis with acquisition times ranging from 30 s to a minute. To the best of our knowledge, this is the first report on the application of DART-MS to the detection and identification of volatile or semi-volatile insect terpenoids, and on the use of DOE approaches to optimize DART-MS analytical procedures.

An improved end-point fluorimetric procedure for the determination of low amounts of trypsin activity in biological samples using rhodamine-110-based substrates.

A novel end-point fluorimetric procedure based on the use of rhodamine-110-labeled specific substrate was developed to determine trypsin activities in biological samples. We evaluated the ability of trichloroacetic acid and acetic acid to stop the enzymatic reaction without hindering the detection of the fluorescence of rhodamine-110 released into the reaction mixture from the specific substrate (CBZ-L-alanyl-L-arginine)(2)-rhodamine-110. Trichloroacetic acid decreased markedly the fluorescence of rhodamine-110, even at low concentrations. On the other hand, the addition of 50 mmol/l acetic acid inactivated efficiently trypsin activity, causing minor effects on rhodamine-110 fluorescence. The proposed procedure was more sensitive than the spectrophotometric end-point method using N-alpha-benzoyl-DL-arginine-p-nitroanilide as substrate. The possibility of carrying out end-point fluorimetric assays improves the performance of monocell fluorimeters by setting specific conditions optimal for each enzyme activity independently of the fluorimeter. This method also allows replicate assays to be conducted simultaneously, resulting in considerable time saving and in increased performance of low-cost equipment.

Allatostatin-C receptors in mosquitoes.

In the present work we describe the functional and molecular characterization of two Aedes aegypti allatostatin-C receptor paralogs (AeAS-CrA and AeAS-CrB) and provide a detailed quantitative study of the expression of the AS-C receptor genes in an adult insect. The tissue distribution of the two AS-C receptors differed significantly; the mRNA levels of AeAS-CrB in the Malpighian tubules were the highest detected, while transcripts for AeAS-CrA were relatively low in this tissue. In addition, the transcript levels of both receptors were different in the thoracic and abdominal ganglia, corpora allata (CA) and the testis of the male. In the CA, the AeAS-CrB mRNA levels were constant from 0 to 72 h after female emergence, while the AeAS-CrA levels increased at 72 h. To complement the receptor expression studies, we analyzed the tissue specificity for allatostatin-C mRNA in female mosquitoes. Expression was high in abdominal ganglia and brain. Transcript levels of allatostatin-C in the head of females were elevated at eclosion and there were no major changes during the first week of adult life or after blood feeding. Fluorometric Imaging Plate Reader (FLIPR) recordings of calcium transients in HEK293T cells transiently expressing both putative receptors showed that they both responded selectively to allatostatin-C stimulation in the nanomolar concentration range. However, the peptide showed slightly greater affinity for AeAS-CrB than AeAS-CrA. Our studies suggest that some of the pleiotropic effects of allatostatin-C in mosquitoes could be mediated by the different receptor paralogs. Transcriptional regulation of the AS-C receptors may not have a critical role in the changes of CA responsiveness to the peptide that we previously described.

NADP+-dependent farnesol dehydrogenase, a corpora allata enzyme involved in juvenile hormone synthesis.

The synthesis of juvenile hormone (JH) is an attractive target for control of insect pests and vectors of disease, but the minute size of the corpora allata (CA), the glands that synthesize JH, has made it difficult to identify important biosynthetic enzymes by classical biochemical approaches. Here, we report identification and characterization of an insect farnesol dehydrogenase (AaSDR-1) that oxidizes farnesol into farnesal, a precursor of JH, in the CA. AaSDR-1 was isolated as an EST in a library of the corpora allata-corpora cardiaca of the mosquito Aedes aegypti. The 245-amino acid protein presents the typical short-chain dehydrogenase (SDR) Rossmann-fold motif for nucleotide binding. This feature, together with other conserved sequence motifs, place AaSDR-1 into the "classical" NADP(+)-dependent cP2 SDR subfamily. The gene is part of a group of highly conserved paralogs that cluster together in the mosquito genome; similar clusters of orthologs were found in other insect species. AaSDR-1 acts as a homodimer and efficiently oxidizes C(10) to C(15) isoprenoid and aliphatic alcohols, showing the highest affinity for the conversion of farnesol into farnesal. Farnesol dehydrogenase activity was not detected in the CA of newly emerged mosquitoes but significant activity was detected 24 h later. Real time PCR experiments revealed that AaSDR-1 mRNA levels were very low in the inactive CA of the newly emerged female, but increased >30-fold 24 h later during the peak of JH synthesis. These results suggest that oxidation of farnesol might be a rate-limiting step in JH III synthesis in adult mosquitoes.

Molecular and functional characterization of a juvenile hormone acid methyltransferase expressed in the corpora allata of mosquitoes.

A juvenile hormone acid methyltransferase (JHAMT) was isolated as an abundant EST in a library of the corpora allata of the adult female mosquito Aedes aegypti. Its full length cDNA encodes a 278-aa protein that has 43% amino acid identity with BmJHAMT, a juvenile hormone acid methyltransferase previously cloned from Bombyx mori. Heterologous expression produced a recombinant protein that metabolizes farnesoic acid (FA) into methyl farnesoate, as well as juvenile hormone acid into juvenile hormone III (JH III) with exquisite stereo specificity. Real time PCR experiments showed that JHAMT mRNA levels are not an unequivocal indicator of JH III synthesis rates; the A. aegypti JHAMT gene, silent in female pupae, was transcriptionally activated just 4-6h before adult eclosion. Radiochemical methyltransferase assays using active and inactive corpora allata glands (CA) dissected from sugar and blood-fed females respectively, clearly indicated that significant levels of JHAMT enzymatic activity are present when the CA shows very low spontaneous rates of JH III synthesis. Having the last enzymes of the JH synthetic pathway readily available all the time might be critical for the adult female mosquito to sustain rapid dynamic changes in JH III synthesis in response to nutritional changes or peripheral influences, such as mating or feeding. These results suggest that this gene has different roles in the regulation of JH synthesis in pupal and adult female mosquitoes, and support the hypothesis that the rate-limiting steps in JH III synthesis in adult female mosquitoes are located before entrance of FA into the synthetic pathway.

Role of juvenile hormone and allatotropin on nutrient allocation, ovarian development and survivorship in mosquitoes.

Teneral reserves are utilized to initiate previtellogenic ovarian development in mosquitoes. Females having emerged with low teneral reserves have reduced juvenile hormone (JH) synthesis and previtellogenic development. We investigated what role JH, allatotropin (AT) and other head-factors play in the regulation of previtellogenic ovarian development and adult survivorship. Factors from the head are essential for corpora allata (CA) activation and reproductive maturation. We have shown that decapitation of females within 9-12h after adult ecdysis prevented normal development of the previtellogenic follicles; however maximum previtellogenic ovarian development could be induced in decapitated females by topically applying a JH analog. When females were decapitated 12 or more hours after emergence nutritional resources had been committed to ovarian development and survivorship was significantly reduced. To study if allatotropin levels correlated with teneral reserves, we measured AT titers in the heads of two adult phenotypes (large and small females) generated by raising larvae under different nutritional diets. In large mosquitoes AT levels increased to a maximum of 45 fmol in day 4; in contrast, the levels of allatotropin in the heads of small mosquitoes remained below 9 fmol during the 7 days evaluated. These results suggest that only when nutrients are appropriate, factors released from the brain induce the CA to synthesize enough JH to activate reproductive maturation.

Biochemical, molecular, and functional characterization of PISCF-allatostatin, a regulator of juvenile hormone biosynthesis in the mosquito Aedes aegypti.

Aedes aegypti PISCF-allatostatin or allatostatin-C (Ae-AS-C) was isolated using a combination of high performance liquid chromatography and enzyme-linked immunosorbent assay (ELISA). The matrix-assisted laser desorption/ionization time-of-flight (TOF) mass spectrum of positive ELISA fractions revealed a molecular mass of 1919.0 Da, in agreement with the sequence qIRYRQCYFNPISCF, with bridged cysteines. This sequence was confirmed by matrix-assisted laser desorption/ionization tandem TOF/TOF mass spectrometry analysis. The corresponding Ae-AS-C cDNA was amplified by PCR, and the sequence of the peptide was confirmed. An in vitro radiochemical assay was used to study the inhibitory effect of synthetic Ae-AS-C on juvenile hormone biosynthesis by the isolated corpora allata (CA) of adult female A. aegypti. The inhibitory action of synthetic Ae-AS-C was dose-dependent; with a maximum at 10(-9) m. Ae-AS-C showed no inhibitory activity in the presence of farnesoic acid, an immediate precursor of juvenile hormone, indicating that the Ae-AS-C target is located before the formation of farnesoic acid in the pathway. The sensitivity of the CA to inhibition by Ae-AS-C in the in vitro assay varied during the adult life; the CA was most sensitive during periods of low synthetic activity. In addition, the levels of Ae-AS-C in the brain were studied using ELISA and reached a maximum at 3 days after eclosion. These studies suggest that Ae-AS-C is an important regulator of CA activity in A. aegypti.