Shirley Luckhart

Molecular characterization of a prophenoloxidase cDNA from the malaria mosquito Anopheles stephensi

Some refractory anopheline mosquitoes are capable of killing Plasmodium, the causative agent of malaria, by melanotic encapsulation of invading ookinetes. Phenoloxidase (PO) appears to be involved in the formation of melanin and toxic metabolites in the surrounding capsule. A cDNA encoding Anopheles stephensi prophenoloxidase (Ans‐proPO) was isolated from a cDNA library screened with an amplimer produced by reverse transcriptase polymerase chain reaction (RT‐PCR) with degenerate primers designed against conserved proPO sequences. The 2.4‐kb‐long cDNA has a 2058 bp open reading frame encoding Ans‐proPO of 686 amino acids. The deduced amino acid sequence shows significant homology to other insect proPO sequences especially at the two putative copper‐binding domains. In A. stephensi, Ans‐proPO expression was detected in larval, pupal and adult stages. The Ans‐proPO mRNA was detected by RT‐PCR and in situ hybridization in haemocytes, fat body and epidermis of adult female mosquitoes. A low level of expression was detected in the ovaries, whereas no expression was detected in the midguts. Semi‐quantitative RT‐PCR analysis of Ans‐proPO mRNA showed that its expression was similar in adult female heads, thoraxes and abdomens. No change in the level of Ans‐proPO expression was found in adult females after blood feeding, bacterial challenge or Plasmodium berghei infection. However, elevated PO activity was detected in P. berghei‐infected mosquitoes, suggesting that in non‐selected permissive mosquitoes PO may be involved in limiting parasite infection. Genomic Southern blot and immunoblots suggest the presence of more than one proPO gene in the A. stephensi genome, which is consistent with the findings in other Diptera and Lepidoptera species. The greatest similarity in sequence and expression profile between Ans‐proPO and A. gambiae proPO6 suggests that they might be homologues. Our results demonstrate that Ans‐proPO is constitutively expressed through different developmental stages and under different physiological conditions, implying that other factors in the proPO activation cascade regulate melanotic encapsulation.

Effects and implications of antibiotic treatment on Wolbachia‐infected vine weevil (Coleoptera: Curculionidae)

1 The parthenogenetic weevil Otiorhynchus sulcatus was collected from five geographical locations in the U.S.A. All weevils from each location were infected by Wolbachia belonging to supergroup B.

The role of As60A, a TGF-β homolog, in Anopheles stephensi innate immunity and defense against Plasmodium infection

We have examined the constitutive and induced expression of As60A in Anopheles stephensi females. As60A is expressed throughout the body of A. stephensi, including the midgut, fat body and developing eggs. We discovered that As60A is induced in the midgut and carcass of A. stephensi in response to Plasmodium infection. Induction of As60A correlates with periods of parasite motility and reproduction. Further, induction is dependent on the intensity of parasite infection: low numbers of parasites do not induce As60A expression. Thus, we conclude that As60A is a component of the A. stephensi immune response to Plasmodium infection. The involvement of a member of the transforming growth factor beta (TGF-beta) super family in the mosquito immune response is analogous to the involvement of TGF-beta1 in the mammalian immune response to Plasmodium. The modulation of As60A and A. stephensi nitric oxide synthase (AsNOS) expression in response to Plasmodium indicates that homologs of effector (NOS) and regulator (TGF-beta1) gene super families may defend evolutionarily diverse hosts against a shared pathogen.

Cross-Talk Between Nitric Oxide and Transforming Growth Factor- β1 in Malaria

Malaria has re-emerged as a global health problem, leading to an increased focus on the cellular and molecular biology of the mosquito Anopheles and the parasite Plasmodium with the goal of identifying novel points of intervention in the parasite life cycle. Anti-parasite defenses mounted by both mammalian hosts and Anopheles can suppress the growth of Plasmodium. Nonetheless, the parasite is able to escape complete elimination in vivo, perhaps by thwarting or co-opting these mechanisms for its own survival, as do numerous other pathogens. Among the defense systems used by the mammalian host against Plasmodium is the synthesis of nitric oxide (NO), catalyzed by an inducible NO synthase (iNOS). Nitric oxide produced by the action of an inducible Anopheles stephensi NO synthase (AsNOS) may be central to the anti-parasitic arsenal of this mosquito. In mammals, iNOS can be modulated by members of the transforming growth factor-beta (TGF-beta) cytokine superfamily. Transforming growth factor-beta is produced as an inactive precursor that is activated following dissociation of certain inhibitory proteins, a process that can be promoted by reaction products of NO as well as by hemin. Ingestion by Anopheles of blood containing Plasmodium initiates parasite development, blood digestion which results in the accumulation of hematin (hemin) in the insect midgut, and induction of both AsNOS and TGF-beta-like (As60A) gene expression in the midgut epithelium. Active mammalian TGF-beta1 can be detected in the A. stephensi midgut up to 48h post-ingestion and latent TGF-beta1 can be activated by midgut components in vitro, a process that is potentiated by NO and that may involve hematin. Further, mammalian TGF-beta1 is perceived as a cytokine by A. stephensi cells in vitro and can alter Plasmodium development in vivo. Bloodfeeding by Anopheles, therefore, results in a juxtaposition of evolutionarily conserved mosquito and mammalian TGF-beta superfamily homologs that may influence transmission dynamics of Plasmodium in endemic regions.