Brenda T. Beerntsen

Genetics of Mosquito Vector Competence

SUMMARY Mosquito-borne diseases are responsible for significant human morbidity and mortality throughout the world. Efforts to control mosquito-borne diseases have been impeded, in part, by the development of drug-resistant parasites, insecticide-resistant mosquitoes, and environmental concerns over the application of insecticides. Therefore, there is a need to develop novel disease control strategies that can complement or replace existing control methods. One such strategy is to generate pathogen-resistant mosquitoes from those that are susceptible. To this end, efforts have focused on isolating and characterizing genes that influence mosquito vector competence. It has been known for over 70 years that there is a genetic basis for the susceptibility of mosquitoes to parasites, but until the advent of powerful molecular biological tools and protocols, it was difficult to assess the interactions of pathogens with their host tissues within the mosquito at a molecular level. Moreover, it has been only recently that the molecular mechanisms responsible for pathogen destruction, such as melanotic encapsulation and immune peptide production, have been investigated. The molecular characterization of genes that influence vector competence is becoming routine, and with the development of the Sindbis virus transducing system, potential antipathogen genes now can be introduced into the mosquito and their effect on parasite development can be assessed in vivo. With the recent successes in the field of mosquito germ line transformation, it seems likely that the generation of a pathogen-resistant mosquito population from a susceptible population soon will become a reality.

Transcriptional Profiling of Midgut Immunity Response and Degeneration in the Wandering Silkworm, Bombyx mori

Background Lepidoptera insects have a novel development process comprising several metamorphic stages during their life cycle compared with vertebrate animals. Unlike most Lepidoptera insects that live on nectar during the adult stage, the Bombyx mori silkworm adults do not eat anything and die after egg-laying. In addition, the midguts of Lepidoptera insects produce antimicrobial proteins during the wandering stage when the larval tissues undergo numerous changes. The exact mechanisms responsible for these phenomena remain unclear. Principal Findings We used the silkworm as a model and performed genome-wide transcriptional profiling of the midgut between the feeding stage and the wandering stage. Many genes concerned with metabolism, digestion, and ion and small molecule transportation were down-regulated during the wandering stage, indicating that the wandering stage midgut loses its normal functions. Microarray profiling, qRT-PCR and western blot proved the production of antimicrobial proteins (peptides) in the midgut during the wandering stage. Different genes of the immune deficiency (Imd) pathway were up-regulated during the wandering stage. However, some key genes belonging to the Toll pathway showed no change in their transcription levels. Unlike butterfly (Pachliopta aristolochiae), the midgut of silkworm moth has a layer of cells, indicating that the development of midgut since the wandering stage is not usual. Cell division in the midgut was observed only for a short time during the wandering stage. However, there was extensive cell apoptosis before pupation. The imbalance of cell division and apoptosis probably drives the continuous degeneration of the midgut in the silkworm since the wandering stage. Conclusions This study provided an insight into the mechanism of the degeneration of the silkworm midgut and the production of innate immunity-related proteins during the wandering stage. The imbalance of cell division and apoptosis induces irreversible degeneration of the midgut. The Imd pathway probably regulates the production of antimicrobial peptides in the midgut during the wandering stage.

A systematic study on hemocyte identification and plasma prophenoloxidase from Culex pipiens quinquefasciatus at different developmental stages.

Culexpipiens quinquefasciatus (C. quinquefasciatus) is an important vector that can transmit human diseases such as West Nile virus, lymphatic filariasis, Japanese encephalitis and St. Louis encephalitis. However, very limited research concerning the humoral and cellular immune defenses of C. quinquefasciatus has been done. Here we present the research on hemocyte identification and plasma including hemocyte prophenoloxidase from C. quinquefasciatus at all developmental stages in order to obtain a complete picture of C. quinquefasciatus innate immunity. We identified hemocytes into four types: prohemocytes, oenocytoids, plasmatocytes and granulocytes. Prophenoloxidase (PPO) is an essential enzyme to induce melanization after encapsulation. PPO-positive hemocytes and plasma PPO were observed at all developmental stages. As for specific hemocyte types, prophenoloxidase was found in the plasmatocytes at larval stage alone and in the smallest prohemocytes during almost all developmental stages. Moreover, the granulocytes were PPO-positive from blood-fed female mosquitoes and oenocytoids were observed PPO-positive in pupae and in adult females after blood-feeding. As for plasma, there were different patterns of PPO in C. quinquefasciatus at different developmental stages. These results are forming a basis for further studies on the function of C. quinquefasciatus hemocytes and prophenoloxidase as well as their involvement in fighting against mosquito-borne pathogens.

Properties of Drosophila melanogaster prophenoloxidases expressed in Escherichia coli.

Insect prophenoloxidases (PPOs) are a group of important innate immunity proteins. Although there have been numerous studies dealing with the PPO activation cascade, the detailed biochemical behaviors of the PPO family proteins remain to be clearly established. This is due primarily to the difficulty in obtaining adequate amounts of PPO proteins for comprehensive characterization. In this study, we expressed three Drosophila melanogaster PPO genes in Escherichia coli, and extensively evaluated expression conditions for obtaining soluble proteins. Through the manipulation of expression conditions, particularly the culture temperature of PPO-transformed E. coli cells, we were able to obtain large quantities of soluble recombinant PPO proteins. Additional Cu(2+), either added into the culture medium during PPO induction or directly mixed with the purified rPPO preparations, was necessary to produce Cu(2+) associated proenzymes. Cu(2+) associated PPOs showed obvious enzyme activities after activation by either ethanol or cetylpyridinium chloride, or by AMM1 (a pupal protein fraction containing native serine proteases for PPO activation). Dose responses for association of individual purified Drosophila rPPOs with Cu(2+) showed that Drosophila rPPO1 and rPPO3 had relatively higher affinity for Cu(2+) than rPPO2 did. Surprisingly, however, high concentration of Cu(2+) (2 mM) completely inhibited PPO activity. Each rPPO had similar activity when dopamine or l-DOPA was the substrate. However, rPPO1 alone had very high activity if l-tyrosine was used as a substrate. After activation by ethanol or 2-propanol, Km and Vmax of the three rPPOs changed as shown in the following: rPPO2<rPPO3<rPPO1. If activated by ethanol, the Km and Vmax of each rPPO were lower than by 2-propanol. Due to the difficulty in obtaining functional PPOs via traditional purification methods, the method established in this study will be helpful to produce active insect recombinant PPOs for the study of PPO properties and functions in the future.

BmToll9, an Arthropod conservative Toll, is likely involved in the local gut immune response in the silkworm, Bombyx mori.

The Toll family of transmembrane proteins mediates signaling during the innate immune response in most animals. Toll9 is widespread in insects and has a unique signature, QHR, in its Toll/interleukin-1 receptor (TIR) domain. The introns in the TIR region are highly conserved among insects, suggesting the antiquity of Toll9 genes. Toll9 of Bombyx mori (BmToll9) was analysed by quantitative real-time RT-PCR. BmToll9 is constitutively expressed in egg, larval and adult stages prior to microbial challenge. BmToll9 is strongly expressed in the different parts of the gut, but weakly expressed in haemocytes, trachea, fat body, malpighian tubule and epidermis, and scarcely expressed in the silk glands. The injection of sterilized 0.85% NaCl solution inhibited BmToll9 expression in most tissues especially during the early responses. Staphylococcus aureus had no or limited effect on the expression of BmToll9 in the silkworm gut and fat body. But in epidermis, trachea, malpighian tubules and haemocytes, the expression of BmToll9 was significantly increased after S. aureus challenge. Infection of Escherichia coli significantly increased the BmToll9 expression in different parts of the gut as well as in epidermis, malpighian tubule and haemocytes. At 48h after feeding of the fungus, Beauveria bassiana, BmToll9 expression was significantly increased. Tissues responses to the injected and ingested bacteria showed that BmToll9 is probably involved in the local gut immune response in the silkworm.

Global identification of bursicon-regulated genes in Drosophila melanogaster

BackgroundBursicon is a heterodimer neuropeptide responsible for regulating cuticle sclerotization and wing expansion in several insect species. Recent studies indicate that the action of bursicon is mediated by a specific G protein-coupled receptor DLGR2 and the cAMP/PKA signaling pathway. However, little is known regarding the genes that are regulated by bursicon. The identification of bursicon-regulated genes is the focus of this investigation.ResultsWe used DNA microarray analysis to identify bursicon-regulated genes in neck-ligated flies (Drosophila melanogaster) that received recombinant bursicon (r-bursicon). Fifty four genes were found to be regulated by bursicon 1 h post r-bursicon injection, 52 being up-regulated and 2 down-regulated while 33 genes were influenced by r-bursicon 3 h post-injection (24 up-regulated and 9 down-regulated genes). Analysis of these genes by inference from the fly database http://flybase.bio.indiana.edu revealed that these genes encode proteins with diverse functions, including cell signaling, gene transcription, DNA/RNA binding, ion trafficking, proteolysis-peptidolysis, metabolism, cytoskeleton formation, immune response and cell-adhesion. Twenty eight genes randomly selected from the microarray-identified list were verified by real time PCR (qPCR) which supported the microarray data. Temporal response studies of 13 identified and verified genes by qPCR revealed that the temporal expression patterns of these genes are consistent with the microarray data.ConclusionUsing r-bursicon, we identified 87 genes that are regulated by bursicon, 30 of which have no previously known function. Most importantly, all genes randomly selected from the microarray-identified list were verified by real time PCR. Temporal analysis of 13 verified genes revealed that the expression of these genes was indeed induced by bursicon and correlated well with the cuticle sclerotization process. The composite data suggest that these genes play important roles in regulating the cuticle sclerotization and wing expansion processes. The data obtained here will form the basis for future studies aimed at elucidating the exact mechanisms upstream from the secretion of bursicon and its binding to target cells.

Drosophila melanogaster prophenoloxidases respond inconsistently to Cu2+ and have different activity in vitro

Dipteran insects, like mosquitoes, possess more than two prophenoloxidase (PPO) genes, but it is unclear whether their gene products differ in biochemical properties and physiological functions. Here, we used three Drosophila melanogaster PPOs as models to study their properties through expression in S2 cells. Our data revealed that the PPOs were expressed in the ethanol-activatable conformation: rPPO1 and rPPO2 needed additional Cu(2+) in the medium, but rPPO3 did not. rPPO1 bound Cu(2+) within minutes; rPPO2 did that in hours when Cu(2+) were present at a higher concentration. Thus, rPPO1 and rPPO2 were expressed as apo-rPPO and became holo-PPO upon Cu(2+) binding; rPPO3 was holo-PPO immediately after expression. Surprisingly, in the absence of ethanol, the apparently intact rPPO3 catalyzed dopamine oxidation and melanization. The successful method for rPPO expression in S2 cells described in this paper will provide us with an opportunity to study the properties of a specific PPO gene in a small insect like mosquitoes in the future.

Hemocyte alterations during melanotic encapsulation of Brugia malayi in the mosquito Armigeres subalbatus.

The involvement of hemocytes in melanotic encapsulation reactions against Brugia malayi was assessed in Armigeres subalbatus. Hemocyte populations, epitope changes, phenol oxidase (PO) activity, and the presence of an 84-kDa polypeptide were investigated in mosquitoes exposed to a B. malayi-infective bloodmeal (= immune-activated), in mosquitoes given a noninfective bloodmeal (= controls), in nonbloodfed mosquitoes (= naive), or in some combination of these. Total hemocyte populations in immune-activated mosquitoes significantly decreased at 24 hr postbloodmeal (PB) as compared with controls. At 48 and 72 hr PB, hemocyte population levels in immune-activated mosquitoes increased to control levels. Epitope changes, as indicated by wheat germ agglutinin (WGA) binding, also were observed. There was a significant increase in the percentage of hemocytes binding WGA in immune-activated mosquitoes at 24 hr PB as compared with controls. Furthermore, the activity of hemocyte PO, an enzyme involved in the melanotic encapsulation pathway, was significantly elevated at 12 hr PB in immune-activated mosquitoes as compared with controls. Analysis for the presence of an 84-kDa polypeptide in A. subalbatus indicates that a 2.0-kb message in total RNA hybridized to D6.12, an Aedes aegypti cDNA encoding an 84-kDa polypeptide that is associated with melanotic encapsulation responses. The hybridization of D6.12 to RNA was not greater in immune-activated as compared to control A. subalbatus, as has been observed in A. aegypti. Results indicate that these hemocyte changes correspond in time with the melanotic encapsulation reactions of A. subalbatus against filarial worms.

Photoacoustic spectroscopy of β-hematin.

Malaria affects over 200 million individuals annually, resulting in 800,000 fatalities. Current tests use blood smears and can only detect the disease when 0.1-1% of blood cells are infected. We are investigating the use of photoacoustic flowmetry to sense as few as one infected cell among 10 million or more normal blood cells, thus diagnosing infection before patients become symptomatic. Photoacoustic flowmetry is similar to conventional flow cytometry, except that rare cells are targeted by nanosecond laser pulses to induce ultrasonic responses. This system has been used to detect single melanoma cells in 10 ml of blood. Our objective is to apply photoacoustic flowmetry to detection of the malaria pigment hemozoin, which is a byproduct of parasite-digested hemoglobin in the blood. However, hemozoin is difficult to purify in quantities greater than a milligram, so a synthetic analog, known as β-hematin was derived from porcine haemin. The specific purpose of this study is to establish the efficacy of using β-hematin, rather than hemozoin, for photoacoustic measurements. We characterized β-hematin using UV-vis spectroscopy, TEM, and FTIR, then tested the effects of laser irradiation on the synthetic product. We finally determined its absorption spectrum using photoacoustic excitation. UV-vis spectroscopy verified that β-hematin was distinctly different from its precursor. TEM analysis confirmed its previously established nanorod shape, and comparison of the FTIR results with published spectroscopy data showed that our product had the distinctive absorbance peaks at 1661 and 1206 cm(-1). Also, our research indicated that prolonged irradiation dramatically alters the physical and optical properties of the β-hematin, resulting in increased absorption at shorter wavelengths. Nevertheless, the photoacoustic absorption spectrum mimicked that generated by UV-vis spectroscopy, which confirms the accuracy of the photoacoustic method and strongly suggests that photoacoustic flowmetry may be used as a tool for diagnosis of malaria infection.

Specific amino acids affecting Drosophila melanogaster prophenoloxidase activity in vitro

Insect prophenoloxidase (PPO) is a key enzyme that induces melanization around invading pathogens and at wounds to prevent further infection. Drosophila melanogaster has three PPO genes which have different biochemical properties following over-expression in S2 cells. As shown by automatic melanization of S2 cells, recombinant PPO3 (rPPO3) became activated upon Cu(2+) addition (Cu(2+)-aided cells melanization without ethanol activation and substrate addition: +Cu(2+); -DOPA, -Ethanol). The exact reasons for this phenomenon are still unknown. In this study, using site-directed mutagenesis and over-expression methods, we found that the place holder, two independent amino acids (equal to Manduca sexta amino acid residues: F218 and S393 in MsPPO1, F224 and E395 in MsPPO2) in the active site pocket and a missing fragment (similar to (565)RPGDPGT(571) in MsPPO1 and (571)QGSDPRR(577) in MsPPO2) at the C-terminus of PPO3, affect rPPO3-S2 cells Cu(2+)-aided auto-melanization. Some mutations nearly rescued rPPO3 Cu(2+)-aided auto-activation, which suggests that the auto-activation of wild type rPPO3 was not due to cleavage by serine proteases. We also found that the corresponding amino acids in the active site pocket have similar effect on PPO1 as on PPO3. PPO1 staining activity (Cu(2+) added or not during PPO transfection; cells melanized after ethanol activation and substrate addition: ±Cu(2+); +DOPA, +Ethanol) has a positive relationship with the active site pocket size as does rPPO3. The fragment of rPPO1 corresponding to the one missing from the C-terminus of PPO3 has no influence on rPPO1 staining activity after it is deleted. However, the staining activities of rPPO2 mutants decreased after deletion of those corresponding amino acid sequences. When the corresponding fragments from PPO1 or PPO2 were inserted into PPO3, the mutant rPPO3 had no influence on staining activity, but had a significantly lowered Cu(2+)-aided auto-activation. Thus, we found that some amino acids are important for rPPO3 Cu(2+)-aided auto-activation as well as PPO staining activity in vitro.