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Immune Activation by Life-Shortening Wolbachia and Reduced Filarial Competence in Mosquitoes

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Science  02 Oct 2009:
Vol. 326, Issue 5949, pp. 134-136
DOI: 10.1126/science.1177531

Mosquito Vector Intervention

Mosquitoes are responsible for causing the infection of an estimated 120 million people with the nematode worms that block the lymph system and result in the gross pathology of elephantiasis and other filariases. Kambris et al. (p. 134) infected vector mosquitoes with a bacterium (Wolbachia), which impaired the insects' ability to act as filaria vectors and possibly would affect transmission of other pathogens, too. The infected mosquitoes were less susceptible to the worms owing to chronic upregulation of mosquito immune responses. Immune activation bears a physiological cost for the mosquitoes, which may explain earlier observations of curtailed life spans of Wolbachia-infected mosquitoes.

Abstract

Wolbachia strain wMelPop reduces the longevity of its Drosophila melanogaster host and, when introduced into the mosquito Aedes aegypti, halves its life span. We show that wMelPop induces up-regulation of the mosquito’s innate immune system and that its presence inhibits the development of filarial nematodes in the mosquito. These data suggest that wMelPop could be used in the global effort to eliminate lymphatic filariasis and possibly for the control of other mosquito-borne parasites where immune preactivation inhibits their development. The cost of constitutive immune up-regulation may contribute to the life-shortening phenotype.

Wolbachia pipientis is a maternally inherited intracellular bacterium of invertebrates, capable of spreading itself through populations by reproductive manipulation such as cytoplasmic incompatibility (CI). The strain wMelPop or “popcorn,” unusually, reduces the longevity of its Drosophila melanogaster host (1) and also has been shown to halve life-span when the mosquito Aedes aegypti was stably transinfected (2). The wMelPop life-shortening phenotype offers the prospect of a disease control system by potentially skewing the population structure toward younger individuals. Vectorial capacity is particularly sensitive to mosquito age because mosquito-borne pathogens require an extrinsic incubation period between ingestion and transmission that is long relative to mean life-span in the field, such that only older mosquitoes within a population are potentially infective. wMelPop was also found to be inherited at high rates and to induce strong CI in Ae. aegypti, which provides a reproductive advantage to infected females. The wMelPop strain should be capable of spreading through populations despite the reduction in mean life-span, because reproduction by older individuals makes a relatively small contribution to the next generation (26).

We compared host gene expression using whole-genome microarrays in genetically identical Ae. aegypti lines infected and uninfected with wMelPop (7) to examine the mechanism underlying the life-shortening phenotype. Of 199 gene transcripts up-regulated by more than a twofold threshold, 78 had putative immune-related functions (Fig. 1 and table S2). These included genes that encode 17 CLIP domain, serine proteases, nine FREPs (fibrinogen-related proteins), six cecropins, four TEPs (thioester-containing proteins), three defensins, three PPOs (prophenoloxidases), two lysozymes, two PGRPs (peptidoglycan recognition proteins), two GNBPs (Gram-negative binding proteins), and the nuclear transcription factor Rel2. Various effector genes, particularly cecropins and other antimicrobial peptides, show the highest up-regulation (Fig. 1 and table S2). Five immune-related genes (primarily of predicted regulatory function) were down-regulated below the twofold threshold (table S2).

Fig. 1

Induction of immune gene transcription in wMelPop-infected female Ae. aegypti. A summary of data from four replicate Ae. aegypti microarrays is shown as gene transcripts significantly up-regulated in wMelPop-infected versus uninfected female mosquitoes. Of 199 up-regulated gene transcripts with fold change (FC) ≥ 2 (P ≤ 0.05), a considerable proportion had a putative immune function (immunity) compared with genes of other known function (other) and unknown function (unknown). Gene transcripts with immune-related function were categorized into effectors, signal modulation, and pattern recognition. Gene transcripts encoding immune effectors dominated the list of highly up-regulated genes (FC ≥ 10).

Quantitative RT-PCR (qRT-PCR) experiments with mosquitoes at 2 and 15 days post eclosion using a subset of immune-related genes, provided broad support for the array data (Fig. 2 and table S1). Up-regulation occurred at similar levels at both age points. This scale of constitutive or chronic immune up-regulation is unexpected (the short-lived acute immune gene expression is commonly observed after septic challenge). In Drosophila simulans and Aedes albopictus, naturally occurring Wolbachia was found neither constitutively to induce nor to suppress the transcription of various inducible antibacterial genes (8). Constitutive up-regulation of immune genes is known to have a high cost, and trade-offs between longevity and pathogen resistance associated with immune up-regulation have been described in D. melanogaster (9). Therefore, constitutive up-regulation of immune genes associated with wMelPop infection also provides a possible mechanism for, or contributory factor to, the life-shortening phenotype.

Fig. 2

Microarray and qRT-PCR analyses of differentially regulated genes. The differential regulation of transcript levels in wMelPop-infected Ae. aegypti versus a genetically identical uninfected Ae. aegypti line was examined for a selection of nine up-regulated genes corresponding to two cecropins (CECE, AAEL000611; CECD, AAEL000598), a fibrinogen- or fibronectin-related protein (FREP38, AAEL0015428), a C-type galactose-specific lectin (AAEL005641), a transferrin (AAEL0015458), two CLIP-domain serine proteases (CLIPB46 AAEL005431, CLIPB37 AAEL005093), a peptidoglycan recognition protein (PGRPS1, AAEL009474), a thioester-containing protein (TEP20, AAEL001794), and a conserved hypothetical protein that was down-regulated (CHP, AAEL003467). The values shown are the means (±SEM) of three different qRT-PCR experiments with independent samples, at 2 and 15 days post eclosion.

The development of various pathogens is known to be inhibited by immune preactivation in mosquitoes, which suggests that the constitutive immune regulation observed could influence the transmission of these pathogens to humans. For example, when Ae. aegypti was injected with bacteria 24 hours before challenge with Brugia filarial nematodes, causative agents of human lymphatic filariasis, prevalence and mean intensity of infection were reduced significantly (10). Synthetic cecropin peptides also have known filarial-killing activity (11); data from the microarray experiment showed that six cecropin genes were all up-regulated by more than 25-fold in the presence of wMelPop (table S2).

We investigated the effect of wMelPop on filarial transmission. A filarial susceptibility locus from the susceptible Refm strain of Ae. aegypti was backcrossed into the wMelPop-infected and control uninfected backgrounds by using a visible marker linked to the susceptibility locus (7). A blood meal infected with Brugia pahangi microfilariae (a rodent filarial nematode previously shown to be a good model for studies of the genetics of susceptibility to infection by human filariae) was provided (12). In comparison with the uninfected control line, significant reductions in the mean numbers of third larval stage (L3) infective worms, as well as in the prevalence of infected mosquitoes, were observed at three microfilarial densities when the wMelPop infection was present (Fig. 3). The inhibition effect was strongest at the lowest microfilarial density used, 11 microfilariae per microliter of blood, with 79.8% (A bars) or 84.2% (B bars) reductions in mean L3 stage larvae in the wMelPop-infected line compared with the wMelPop-uninfected control. All microfilarial densities used are high compared with blood densities that would occur naturally.

Fig. 3

wMelPop-infected mosquitoes show a reduction in filarial infection prevalence and intensity. The mean numbers (±SEM) of infective third (L3) stage Brugia pahangi larvae are shown at 10 to 13 days post microfilarial challenge in Ae. aegypti wMelPop-infected (Ae_Pop) versus uninfected (Ae_Tet) lines, that are filaria-susceptible after backcrossing with the Refm strain. Four independent challenge experiments are shown with microfilarial densities (microfilariae per microliter blood) of 11 (experiments A and B), 13 (C), and 23 (D). Values above bars show the prevalence of filarial infection as a proportion of mosquitoes that contained at least one L3 Brugia larva versus the total number of mosquitoes dissected in each category. Differences were significant at *P < 0.01 or **P < 0.001 (Mann-Whitney U test).

Filarial nematodes are somewhat harmful to mosquitoes. To investigate whether the presence of wMelPop conferred protective effects against other insect pathogens, the two lines were challenged by thoracic pricking with a virulent strain of the Gram-negative bacterium Erwinia carotovora. There was higher survival when wMelPop was present than in the wMelPop-free control (Fig. 4). A control challenge with the Gram-positive bacterium Micrococcus luteus confirmed that the high mortality seen when the wMelPop-uninfected line was challenged with Erwinia was not simply a result of septic injury.

Fig. 4

Increased resistance to a pathogenic bacterium in wMelPop-infected mosquitoes. The daily survival rates (%) of Ae. aegypti carrying wMelPop (Ae_Pop) were compared with the Wolbachia-free line (Ae_Tet) after infection with E. carotovora 15 (Ecc15), a pathogenic Gram-negative bacterium. The horizontal axis represents the incubation time after infection in hours. Control infections with the Gram-positive M. luteus demonstrate that the early death or protection effects are not due to septic wounding alone.

Any protective effects provided against harmful pathogens commonly encountered by mosquitoes will act to increase the population-spreading capacity of the wMelPop strain. However, any benefits accrued from pathogen protection in nature are likely only partially to offset the fitness costs associated with immune gene expression. The immune activation phenotype may be a side effect of the unusually fast replication of wMelPop and/or because the immune evasion strategies normally used by Wolbachia are impaired in this novel host. wMelPop Wolbachia must itself be at least partly resistant to the immune effectors induced, because it is maternally transmitted at high levels in infected Ae. aegypti (2).

Wolbachia infections, including wMelPop, were recently reported to provide a protective function against pathogenic viruses in Drosophila (13, 14). The up-regulation of immune genes provides a possible explanation or contributory factor, because there is a degree of overlap between immune peptides induced by bacteria and viruses (15, 16). Cecropins have previously been shown to have antiviral effects, for example, inhibiting HIV-1 (human immunodeficiency virus 1) replication (17), and an artificial cecropin-defensin hybrid peptide inhibited dengue virus replication (18). Knockdown studies will allow these hypotheses to be tested. Cecropins are also known to have a major inhibitory effect on Plasmodium development (19, 20). Furthermore, the orthologs of Rel2, the TEP20 (gene identifier AAEL001794) (7), and LRIM1 (AAEL012086), all up-regulated in the presence of wMelPop, have been shown by knockdown studies in Anopheles to regulate the intensity of Plasmodium infection (2126), as has mosquito midgut microbiota by means of immune stimulation (27). wMelPop-transinfected Anopheles species should be tested for their ability to inhibit transmission of Plasmodium in order to evaluate this malaria control strategy.

Around 120 million people across the tropics are infected with the filarial nematode species that cause lymphatic filariasis, a leading global cause of disability. Ae. aegypti is not a natural vector of the disease; however, other Aedes species are primary regional vectors, such as Ae. polynesiensis in the South Pacific region. The need for novel control methods for Ae. polynesiensis has been previously highlighted (28). Sustained efforts to eliminate filariasis in the region by mass drug administration alone have failed; this is thought to be because Ae. polynesiensis is such an efficient vector of Wuchereria bancrofti, the main causative agent of human lymphatic filariasis, at low microfilarial densities. Another attractive target is Culex quinquefasciatus, the major urban vector of W. bancrofti across the tropics. Once stable wMelPop infections are created, they would need to be challenged directly with W. bancrofti, because it remains possible that the effects observed are species-specific, but the prospect of a self-spreading transmission-reduction system is attractive, particularly given that it is likely to be especially effective when microfilarial densities in the human population are low.

The data reported here suggest wMelPop may represent a valuable tool in the global effort to eradicate lymphatic filariasis and possibly for the control of other mosquito-borne parasites. The combination of direct inhibitory effects on filariae in the mosquito, together with life-span shortening, could have a powerful overall impact in reducing disease transmission. The ability of wMelPop, in common with many other Wolbachia strains, to drive itself through populations using CI makes this a very attractive strategy.

Supporting Online Material

www.sciencemag.org/cgi/content/full/326/5949/134/DC1

Materials and Methods

Tables S1 and S2

References

References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. This study was supported by the Wellcome Trust, European Union’s Framework Programme F7 award 223241, European Molecular Biology Organization, and University of Oxford John Fell Fund. We thank S. O’Neill for providing us with the wMelPop-infected and cured Ae. aegypti lines PGYP1 and PGYP1.tet; to E. Devaney, for providing Brugia pahangi and Refm Ae. aegypti; to M. Povelones, for assistance identifying Ae. aegypti LRIM genes; and to A. Brown for comments on the manuscript. Microarray data have been submitted to the National Center for Biotechnology Information Gene Expression Omnibus database, accession GSE17469.
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