Salvador Hernández-Martínez

Cellular-Mediated Reactions to Foreign Organisms Inoculated into the Hemocoel of Anopheles albimanus (Diptera: Culicidae)

Abstract The immune response against different organisms and particles inoculated in the hemocoel of female Anopheles albimanus Wiedemann was investigated. Histological and ultrastructural observations indicated that melanization and hemocyte type participation varied according to the particles inoculated. The initial responses against heat-killed Microccocus lysodeikticus and Escherichia coli included hemocyte lysis and melanization whereas the response to heat-killed Saccharomyces cerevisiae was only cellular, and an initial melanization of Sephadex G-25 (neutral charged) beads was followed by the formation of cellular aggregates. After 24 h, hemocytes were involved in all terminal encapsulation events. Plasmodium vivax Grassi and Feletti formalin-fixed sporozoites induced a weak response. Cellular aggregates were observed 1 h postinoculation, but participating hemocytes could not be identified because of the extensive cellular damage and lysis. Sporozoites were also observed in the core of these aggregates, mixed with cell debris and free in the hemolymph. The effect on the inoculated particles was also different—S. cerevisiae was encapsulated only by hemocytes, whereas M. lysodeikticus was lysed and E. coli was phagocytosed by plasmatocytes. These results indicate that hemocytes are important components in the immune response in An. albimanus.

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.

Immunostaining for allatotropin and allatostatin-A and -C in the mosquitoes Aedes aegypti and Anopheles albimanus

Confocal laser-scanning microscopy was used to carry out a comparative study of the immunostaining for three families of neuropeptides, viz., allatostatin-A (AS-A), allatostatin-C (AS-C) and allatotropin (AT), in adult female mosquitoes of Aedes aegypti and Anopheles albimanus. The specific patterns of immunostaining for each of the three peptides were similar in both species. The antisera raised against AT, AS-A, and AS-C revealed intense immunoreactivity in the cells of each protocerebral lobe of the brain and stained cells in each of the ventral ganglia and neuronal projections innervating various thoracic and abdominal tissues. Only the AS-A antiserum labeled immunoreactive endocrine cells in the midgut. The distribution of the peptides supports the concept that they play multiple regulatory roles in both species.

SUPEROXIDE ANION IN ANOPHELES ALBIMANUS HEMOLYMPH AND MIDGUT IS TOXIC TO PLASMODIUM BERGHEI OOKINETES

The mechanisms of Plasmodium spp. elimination in resistant mosquitoes are not completely understood. Some resistant anopheline strains are able to melanize Plasmodium spp. ookinetes in their midguts. Because quinoid compounds are potent catalysts for free radical generation and because these radicals can be generated in association with melanogenesis, it is probable that they play an important role in the elimination of parasites. The production of the superoxide anion (O−2) in the hemolymph and midgut of Anopheles albimanus female mosquitoes and its cytotoxic effect on Plasmodium berghei ookinetes were analyzed. Ookinetes inoculated into the hemocoel of A. albimanus were covered with melanin and then encapsulated by hemocytes within 1 hr. The presence of O−2 in midguts and in hemolymph obtained by perfusion was verified by the reduction of 3-(4,5 dimethylthiazolil-2)-2,5-diphenyl tetrazolium bromide. O−2 was generated in the hemolymph obtained by perfusion and midguts only in the presence of dihydroxyphenylalanine (l-DOPA), and this reaction was inhibited by superoxide dismutase (SOD). Plasmodium berghei ookinetes exposed to hemolymph plus l-DOPA were killed in vitro, but addition of SOD prevented their killing. Prophenoloxidase transcripts were not observed in midgut epithelium, suggesting that toxic compounds may be imported from the hemolymph. These results suggest that A. albimanus hemolymph and midguts produce O−2 that may limit Plasmodium spp. parasite development.

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.

Inhibition of juvenile hormone biosynthesis in mosquitoes: effect of allatostatic head factors, PISCF- and YXFGL-amide-allatostatins.

We investigated the role of head factors and allatostatins (ASs) on the regulation of juvenile hormone (JH) synthesis in female adult mosquito. The biosynthetic activity of the Aedes aegypti corpora allata (CA) in vitro was inhibited by factors present in the head. Disconnecting the CA from the brain resulted in a significant increase in the rate of JH biosynthesis. Inhibition was not dependent on intact nervous connections; co-incubation of CA with brains or brain extracts resulted in a significant decrease of JH biosynthesis. This inhibitory effect of brain extracts was reversible and heat stable; extracts lost the inhibitory activity after proteinase K digestion suggesting a peptidic structure. In a first attempt to elucidate the nature of this inhibitory factor, we tested in our CA in vitro system the effect of members of two families of allatostatins already described in mosquitoes. Anopheles gambiae PISCF-allatostatin (homolog to Manduca PISCF-allatostatin) significantly inhibited JH synthesis, while Ae. aegypti YXFGL-amide-allatostatins (homologs to cockroach YXFGL-amide-allatostatins) did not affect JH synthesis. These results represent the first description of an allatostatic effect of PISCF-allatostatins outside the Lepidoptera.

Cloning and epitope mapping of Cry11Aa-binding sites in the Cry11Aa-receptor alkaline phosphatase from Aedes aegypti.

Cry11Aa is the most active Bacillus thuringiensis israelensis toxin against Aedes aegypti larvae. Ae. aegypti alkaline phosphatase (ALP) was previously identified as a Cry11Aa receptor mediating toxicity. Here we report the cloning and functional characterization of this Ae. aegypti Cry11Aa-ALP receptor. Of three ALPs cDNA clones, the recombinant produced ALP1 isoform was shown to bind Cry11Aa and P1.BBMV peptide phage that specifically binds the midgut ALP-Cry11Aa receptor. An anti-ALP1 antibody inhibited binding to brush border membrane vesicles and toxicity of Cry11Aa in isolated cultured guts. Two ALP1 Cry11Aa binding regions (R59-G102 and N257-I296) were mapped by characterizing binding of Cry11Aa to nine recombinant overlapping peptides covering the ALP1 sequence. Finally, by using a peptide spot array of Cry11Aa domain III and site-directed mutagenesis, we show that the ALP1 R59-G102 region binds Cry11Aa through domain II loop alpha-8 while ALP1 N257-I296 interacts with Cry11Aa through domain III 561RVQSQNSGNN570 located in beta18-beta19. Our results show that Cry11Aa domain II and domain III are involved in the binding with two distinct binding sites in the ALP1 receptor.

OKB, a novel family of brain-gut neuropeptides from insects.

In insects, neuropeptides play a central role in the control of most physiological processes. The knowledge and characterization of new neuropeptide families, is of interest on the fields of Genetics, Genomics, Neurobiology, Endocrinology and Evolution. This knowledge also provides the tools for the design of peptidomimetics, pseudopeptides or small molecules, capable of disrupting the physiological processes regulated by the signaling molecules and their receptors. This is a promising target for a novel generation of insecticides. Using database searches, mass spectrometry and RACE-PCR, we identified a neuropeptide precursor transcript encoding a new family of insect neuropeptides in the hemipteran Rhodnius prolixus. We named this precursor Orcokinin B, because is originated by the alternative splicing of the Orcokinin gen. EST and genomic data suggests that Orcokinin B is expressed in the nervous system and gut from several insect species, with the exception of Drosophila sp. (Diptera) and Acyirthosiphon pisum (Hemiptera). Mass spectrometry and RT-PCR confirmed the expression of Orcokinin B in brain and anterior midgut of R. prolixus. Furthermore, we identified orthologues of this new family of peptides in genomic and EST databases from Arachnids and Crustaceans.

Transcriptome of the adult female malaria mosquito vector Anopheles albimanus

BackgroundHuman Malaria is transmitted by mosquitoes of the genus Anopheles. Transmission is a complex phenomenon involving biological and environmental factors of humans, parasites and mosquitoes. Among more than 500 anopheline species, only a few species from different branches of the mosquito evolutionary tree transmit malaria, suggesting that their vectorial capacity has evolved independently. Anopheles albimanus (subgenus Nyssorhynchus) is an important malaria vector in the Americas. The divergence time between Anopheles gambiae, the main malaria vector in Africa, and the Neotropical vectors has been estimated to be 100 My. To better understand the biological basis of malaria transmission and to develop novel and effective means of vector control, there is a need to explore the mosquito biology beyond the An. gambiae complex.ResultsWe sequenced the transcriptome of the An. albimanus adult female. By combining Sanger, 454 and Illumina sequences from cDNA libraries derived from the midgut, cuticular fat body, dorsal vessel, salivary gland and whole body, we generated a single, high-quality assembly containing 16,669 transcripts, 92% of which mapped to the An. darlingi genome and covered 90% of the core eukaryotic genome. Bidirectional comparisons between the An. gambiae, An. darlingi and An. albimanus predicted proteomes allowed the identification of 3,772 putative orthologs. More than half of the transcripts had a match to proteins in other insect vectors and had an InterPro annotation. We identified several protein families that may be relevant to the study of Plasmodium-mosquito interaction. An open source transcript annotation browser called GDAV (Genome-Delinked Annotation Viewer) was developed to facilitate public access to the data generated by this and future transcriptome projects.ConclusionsWe have explored the adult female transcriptome of one important New World malaria vector, An. albimanus. We identified protein-coding transcripts involved in biological processes that may be relevant to the Plasmodium lifecycle and can serve as the starting point for searching targets for novel control strategies. Our data increase the available genomic information regarding An. albimanus several hundred-fold, and will facilitate molecular research in medical entomology, evolutionary biology, genomics and proteomics of anopheline mosquito vectors. The data reported in this manuscript is accessible to the community via the VectorBase website (http://www.vectorbase.org/Other/AdditionalOrganisms/).

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.