Carl Lowenberger

Innate immune response of Aedes aegypti

Insects are able to protect themselves from invasion by pathogens by a rapid and potent arsenal of inducible immune peptides. This fast, extremely effective response is part of the innate immunity exhibited by all insects and many invertebrates, and shows striking similarities with the innate immune response of vertebrates. In Aedes aegypti invasion of the hemocoel by bacteria elicits the production of defensins, cecropins, a peptide active only against Gram-negative bacteria, and several other peptides that we are now characterizing. However, not all insects utilize the same peptides in the same concentrations, which may reflect the pathogens to which they may have been exposed through evolutionary time. These protective measures we see in mosquitoes are the current state of the evolution of a rapid immune response that has contributed to the success of insects in inhabiting essentially every niche on earth. The molecules involved in the response of Aedes aegypti to pathogens, and the potential role of these peptides against eukaryotic parasites ingested and transmitted by mosquitoes are discussed.

Insect immunity: Isolation of three novel inducible antibacterial defensins from the vector mosquito, Aedes aegypti

The injection of Escherichia coli and Micrococcus luteus into the hemocoel of Aedes aegypti induces a potent antibacterial activity in the hemolymph. We have purified and fully characterized three 40-residue antibacterial peptides from the hemolymph of bacteria-challenged mosquitoes that are absent in naive mosquitoes. The peptides are potently active against Gram-positive bacteria and against one of the Gram-negative bacteria that were tested. The amino acid sequences clearly show that the three peptides are novel isoforms of the insect defensin family of antibacterial peptides. They differ from each other by one or two amino acid residues. We present here the complete amino acid sequences of the three isoforms and the activity spectrum of the predominant Aedes defensin.

Cloning and analysis of a cecropin gene from the malaria vector mosquito, Anopheles gambiae.

Parasites of the genus Plasmodium are transmitted to mammalian hosts by anopheline mosquitoes. Within the insect vector, parasite growth and development are potentially limited by antimicrobial defence molecules. Here, we describe the isolation of cDNA and genomic clones encoding a cecropin antibacterial peptide from the malaria vector mosquito Anopheles gambiae. The locus was mapped to polytene division 1C of the X chromosome. Cecropin RNA was induced by infection with bacteria and Plasmodium. RNA levels varied in different body parts of the adult mosquito. During development, cecropin expression was limited to the early pupal stage. The peptide was purified from both adult mosquitoes and cell culture supernatants. Anopheles gambiae synthetic cecropins displayed activity against Gram‐negative and Gram‐positive bacteria, filamentous fungi and yeasts.

Larvicidal and oviposition-altering activity of monoterpenoids, trans-anithole and rosemary oil to the yellow fever mosquito Aedes aegypti (Diptera: Culicidae).

BACKGROUND Aedes aegypti L. is the major vector of dengue fever and dengue hemorrhagic fever. In an effort to find effective tools for control programs to reduce mosquito populations, the authors assessed the acute toxicities of 14 monoterpenoids, trans-anithole and the essential oil of rosemary against different larval stages of Ae. aegypti. The potential for piperonyl butoxide (PBO) to act as a synergist for these compounds to increase larvicidal activity was also examined, and the oviposition response of gravid Ae. aegypti females to substrates containing these compounds was evaluated in behavioral bioassays. RESULTS Pulegone, thymol, eugenol, trans-anithole, rosemary oil and citronellal showed high larvicidal activity against all larval stages of Ae. aegypti (LC(50) values 10.3-40.8 mg L(-1)). The addition of PBO significantly increased the larvicidal activity of all test compounds (3-250-fold). Eugenol, citronellal, thymol, pulegone, rosemary oil and cymene showed oviposition deterrent and/or repellent activities, while the presence of borneol, camphor and beta-pinene increased the number of eggs laid in test containers. CONCLUSIONS This study quantified the lethal and sublethal effects of several phytochemical compounds against all larval stages of Aedes aegypti, providing information that ultimately may have potential in mosquito control programs through acute toxicity and/or the ability to alter reproductive behaviors.

Antimicrobial activity spectrum, cDNA cloning, and mRNA expression of a newly isolated member of the cecropin family from the mosquito vector Aedes aegypti.

An antimicrobial peptide belonging to the cecropin family was isolated from the hemolymph of bacteria-challenged adult Aedes aegypti. This new peptide, named cecropin A, was purified to homogeneity and fully characterized after cDNA cloning. The 34-residue A. aegypti cecropin A is different from the majority of reported insect cecropins in that it is devoid of a tryptophan residue and C-terminal amidation. The importance of these two structural features on the activity spectrum was investigated using a chemically synthesized peptide. A comparison of the antimicrobial activity spectrum of A. aegypti and Drosophilacecropin A showed a lower activity for the mosquito molecule. A. aegypti cecropin mRNA expression was not detected by Northern blot or reverse transcription-polymerase chain reaction analysis in any immature stage of the mosquito, nor in naı̈ve adults, but it was observed in challenged adults 6 h after bacteria inoculation, and it continued over 7–10 days.

Characterization of a defensin from the sand fly Phlebotomus duboscqi induced by challenge with bacteria or the protozoan parasite Leishmania major.

ABSTRACT Antimicrobial peptides are major components of the innate immune response of epithelial cells. In insect vectors, these peptides may play a role in the control of gut pathogens. We have analyzed antimicrobial peptides produced by the sand fly Phlebotomus duboscqi, after challenge by injected bacteria or feeding with bacteria or the protozoan parasite Leishmania major. A new hemolymph peptide with antimicrobial activity was identified and shown to be a member of the insect defensin family. Interestingly, this defensin exhibits an antiparasitic activity against the promastigote forms of L. major, which reside normally within the sand fly midgut. P. duboscqi defensin could be induced by both hemolymph or gut infections. Defensin mRNA was induced following infection by wild-type L. major, and this induction was much less following infections with L. major knockout mutants that survive poorly in sand flies, due to specific deficiencies in abundant cell surface glycoconjugates containing phosphoglycans (including lipophosphoglycan). The ability of gut pathogens to induce gut as well as fat body expression of defensin raises the possibility that this antimicrobial peptide might play a key role in the development of parasitic infections.

Isolation and characterization of a novel insect defensin from Rhodnius prolixus, a vector of Chagas disease.

An antimicrobial peptide belonging to the defensin family of small cationic peptides associated with innate immunity in insects was isolated from the hemolymph of Rhodnius prolixus, a vector of Chagas disease. This peptide, designated R. prolixus defensin A, was purified and sequenced. The active peptide contains 43 residues and aligns well with other insect defensins. However the pre-pro region of the sequence has little shared identity with other insect defensins. We have identified 3 isoforms of R. prolixus defensin from cDNA clones obtained from RNA isolated from the whole bodies of immune activated insects. Northern analysis and Real-Time Quantitative PCR indicate that there is a very low baseline transcription of this peptide in naïve insects, and that transcription increases significantly in the fat body of immune activated insects. In addition there is a delayed induction of transcription of this peptide in the intestine 24 h post activation suggesting that the midgut/intestine of this species is active in the immune response against pathogens.

Reassessing the role of defensin in the innate immune response of the mosquito, Aedes aegypti.

Defensin is the predominant inducible immune peptide in Aedes aegypti. In spite of its activity against Gram‐positive bacteria in vitro, defensin expression is detected in mosquitoes inoculated with Gram‐positive or negative bacteria, or with filarial worms. Defensin transcription and expression are dependent upon bacterial dose; however, translation is inconsistent with transcription because peptide is detectable only in mosquitoes inoculated with large doses. In vitro translation assays provide further evidence for post‐transcriptional regulation of defensin. Clearance assays show that a majority of bacteria are cleared before defensin is detected. In gene silencing experiments, no significant difference in mortality was observed between defensin‐deficient and control mosquitoes after bacteria inoculation. These studies suggest that defensin may have an alternative function in mosquito immunity.

The insect caspases

Developmental and tissue homeostasis is a delicate balance between cell proliferation and cell death. The activation of caspases, a conserved family of cysteine proteases, is a main event in the initiation and execution of programmed cell death. While caspases have been characterized from many organisms, comparatively little is known about insect caspases. In Drosophila melanogaster, seven caspases have been characterized; three initiators and four effectors. In mosquitoes, several putative caspases have been identified in the genomes of Aedes aegypti and Anopheles gambiae. A small number of caspases have been identified in the Lepidoptera, the flour beetle, Tribolium castaneum, and the pea aphid, Acyrthosiphon pisum. The availability of new insect genome sequences will provide a unique opportunity to examine the caspase family across an evolutionarily diverse phylum and will provide valuable insights into their function and regulation.

Identification and characterization of two novel lysozymes from Rhodnius prolixus, a vector of Chagas disease.

Lysozymes have been described in invertebrates as digestive or immune molecules. We report here the characterization of two novel c-type lysozymes, RpLys-A (EU250274) and RpLys-B (EU250275), isolated from the fat body and digestive tract of immune stimulated Rhodnius prolixus, a major vector of Chagas disease. Transcriptional profiles indicate that the temporal and spatial expression patterns of these two peptides are very different. RpLys-A is expressed predominantly in the midgut after ingestion of Trypanosoma cruzi in a bloodmeal, or after injection of bacteria into the hemocoel. RpLys-B is expressed primarily in the fat body after bacterial injection. Phylogenetic alignments indicate that RpLys-A aligns best with molecules from other hemipterans whose major expression is found in the intestinal tract whereas RpLys-B aligns best with mosquito and tick molecules whose expression is found principally in hemocytes and fat body and whose role has been described as immune-related. These data suggest a differential compartmentalized role of two closely related molecules; one for immunity in the hemocoel and the other for digestion in the midgut.