Multiple immunity pathways are implicated in vector competence, which results in insect host resistance to pathogen infection (for mosquito responses to malarial parasites, see [33
]). An initial study that investigated gene expression responses of An. gambiae
to microbial and malaria challenges by using cDNA microarrays constructed from an EST-clone collection noted that the response to malaria parasites partially overlapped with the response to Gram positive (+
) and Gram−
]. Using a similar array approach the mosquito response to the filarial worm has also been found to involve the induction of a large number of genes functioning in the innate immune pathways shown to respond to microbial challenge, although their role in parasite transmission remains to be confirmed [35
In the case of tsetse flies, trypanosome infections induced tsetse’s innate immune responses that are typically involved in clearance of Gram−
bacteria; i.e. the Immune deficient (Imd) pathway [36
]. Trypanosome infection prevalence increased in flies when the expression of the Imd pathway regulator relish
or the downstream expressed antimicrobial peptide (AMP) effector (attacin
) were downregulated by an RNA interference (RNAi)-based reverse genetic analysis before subjecting flies to parasite infections [36
]. In addition, the AMPs Diptericin and Attacin displayed trypanocidal activity both in vitro
and in vivo
in the tsetse’s midgut [38
To understand the role of Aedes aegypti
mosquito immune responses to dengue virus pathogen transmission, Xi et al.
used high-throughput analysis of gene expression and an RNAi approach, reporting that another innate immune pathway (Toll pathway) regulates viral resistance in mosquitoes [39
]. Interestingly, the same study showed that regulation of genes in this immune pathway was also stimulated by natural gut microbiota. Furthermore, when mosquitoes were reared aseptically (in the absence of their endogenous bacterial flora), dengue virus was present in midguts at 2-fold higher titers compared to wild type mosquitoes, implicating once again the microbial fauna in influencing levels of immune resistance.
Early studies on mosquitoes had noted a positive correlation between midgut microbiota and inhibition of Plasmodium
sporozoite development. This phenomenon was demonstrated independently in two different mosquito vectors of Plasmodium
. In one set of experiments An. stephensi
adults were offered a P. falciparum
gametocyte-enriched blood meal that also contained one of four distinct non-native Gram−
bacteria or two distinct Gram+
species. All of the Gram−
bacteria tested were found to partially or completely inhibit oocyst development within the mosquito host. Conversely, the presence of neither Gram+
bacteria resulted in an inhibitory phenotype [40
]. A similar experiment was performed using aseptic An. albimanus
adults and several bacteria that are found naturally in both wild type laboratory and field-captured populations. In this situation, the number of mosquitoes infected with oocysts, and oocyst density, was significantly lower in mosquitoes that received each of the bacteria separately as compared to controls that received no bacteria [41
]. Although no physiological mechanism was proposed at the time, this study indicated that gut microbes have the potential to reduce the capacity of mosquitoes to vector Plasmodium
. Recently aseptic and septic adult An. gambiae
were fed with Plasmodium
gametocytes and subsequently monitored parasite infection status in each group [27
]. Aseptic mosquitoes displayed an increased susceptibility to Plasmodium
infection. This study also found that co-feeding mosquitoes bacteria and P. falciparum
gametocytes resulted in lower than normal infection levels. While ookinete number was the same in the midgut lumen of both mosquito lines, significantly more oocysts were found in the antibiotic treated aseptic mosquitoes. These results indicated that bacteria affect parasite viability prior to the oocyst formation. This may occur while the ookinete is in the midgut or while invading midgut epithelial cells. Global transcription profiling of septic and aseptic mosquitoes identified a significant subset of immune genes in the septic mosquitoes that were presumably upregulated by the host's microbial flora. These immunity genes included several anti-Plasmodium
]. Both expression and infection analyses suggest that the observed anti-Plasmodium
effect is caused by the mosquito’s antimicrobial immune response, possibly through activation of basal immunity [33
The commensal and obligate microbes of tsetse (Sodalis
, respectively) have also been implicated in trypanosome transmission in tsetse. There has been a correlation observed with the presence of the commensal symbiont Sodalis
and in particular specific Sodalis
genotypes and the presence of trypanosome infections in several natural tsetse populations. Also in the absence of the obligate symbiont Wigglesworthia
in the laboratory, flies have been found to be highly susceptible to parasite infections, as discussed below [44
Microbiota may influence their hosts’ vectorial competence by means of direct interaction with parasites. This may occur through inhibitory bioactivity of secreted enzymes or toxins. Alternatively, microbiota may constrain pathogen development indirectly by inducing activity of the host immune system that in turn can clear the pathogenic microbes. Recent studies in several insect systems indicate that both direct and indirect microbiota-induced phenotypes can affect an insect host’s capacity to transmit pathogens (discussed in Box 2
). In fact, both heritable symbionts and environmentally acquired commensal microfauna have been shown to influence this function (reviewed in [45
]). Below we discuss examples of symbiont produced anti-pathogen products, symbiont induced host anti-pathogen products and host immune priming by resident gut fauna as well as by Wolbachia
Box 2. Manipulating the microbiome to modulate insect host vector competence
Endosymbionts are being investigated for their ability to decrease host vector competence. One promising symbiont-based strategy currently under development is called ‘paratransgenesis’. This procedure involves isolating and genetically modifying symbiotic bacteria to express an anti-pathogen molecule. The recombinant symbionts are then reintroduced into their host, where they subsequently increase host resistance to pathogens. Three disease vector systems where this approach has been applied are described below.
- Tsetse: Tsetse’s commensal symbiont, Sodalis, has been genetically modified in vitro to express a marker gene, and then returned to fertile females where they are subsequently passed on to future generations (reviewed in ). Sodalis exhibits a natural resistance to several trypanocidal molecules, including tsetse antimicrobial peptides. Expression of these molecules by Sodalis can result in parasite resistance in tsetse’s midgut [98–100].
- Triatome bugs: Triatomines that transmit the causative agent of Chagas disease (Trypanosoma cruzi) harbor genetically modifiable, gut-associated symbionts that are horizontally transmitted via copharagy . These symbionts can be cultured and genetically modified [101–103]. Host recolonization with genetically modified symbionts that express anti-parasitic molecules subsequently blocks parasite transmission .
- Mosquito: Anopheline mosquitoes form stable associations with bacteria from the genus Asaia that may be used for paratransgenesis . Stable infections can be established in multiple epidemiologically relevant tissues when female mosquitoes were reconstituted with recombinant bacteria [28, 105].
Research is currently under way to improve the efficiency of paratransgenesis by optimizing several variables. These include (i) screening for other commensal symbionts that are stably associated with insect disease vectors, (ii) identifying novel anti-pathogen effector molecules that can be expressed in symbionts, (iii) engineering expression constructs that encode efficacious promoters and secretion signals, and (iv) establishing a mechanism to drive paratransgenic bacteria into field-based insect populations.