Mosquito phenoloxidase and defensin colocalize in melanization innate immune responses.

Mosquitoes mount strong humoral and cellular immune responses against foreign organisms. Two components of the mosquito immune response that have received much attention are the phenoloxidase cascade that leads to melanization and antimicrobial peptides. The purpose of the current study was to use immunocytochemistry and transmission electron microscopy to identify the location of the melanization rate-limiting enzyme phenoloxidase and the antimicrobial peptide defensin in innate immune reactions against Escherichia coli and Micrococcus luteus by the mosquito Aedes aegypti. Our results show that both phenoloxidase and defensin are present at the sites of melanin biosynthesis in immune reactions against bacteria. Furthermore, both proteins are often present inside the same melanotic capsules. When hemocytes were analyzed, phenoloxidase was present in the cytosol of oenocytoids, but no significant amounts of defensin were detected inside any hemocytes. In summary, these data show that phenoloxidase and defensin colocalize in melanization reactions against bacteria and argue for further studies into the potential role of defensin in phenoloxidase-based melanization innate immune responses in mosquitoes.     

Rapid phagocytosis and melanization of bacteria and Plasmodium sporozoites by hemocytes of the mosquito Aedes aegypti

Mosquitoes are vectors of many deadly and debilitating pathogens. In the current study, we used light and electron microscopies to study the immune response of Aedes aegypti hemocytes to bacterial inoculations, Plasmodium gallinaceum natural infections, and latex bead injections. After challenge, mosquitoes mounted strong phagocytic and melanization responses. Granulocytes phagocytosed bacteria singly or pooled them inside large membrane-delimited vesicles. Phagocytosis of bacteria, Plasmodium sporozoites, and latex beads was extensive; we estimated that individual granulocytes have the capacity to phagocytose hundreds of bacteria and thousands of latex particles. Oenocytoids were also seen to internalize bacteria and latex particles, although infrequently and with low capacity. Besides phagocytosis, mosquitoes cleared bacteria and sporozoites by melanization. Interestingly, the immune response toward 2 species of bacteria was different; most Escherichia coli were phagocytosed, but most Micrococcus luteus were melanized. Similar to E. coli, most Plasmodium sporozoites were phagocytosed. The immune response was rapid; phagocytosis and melanization of bacteria began as early as 5 min after inoculation. The magnitude and speed of the cellular response suggest that hemocytes, acting in concert with the humoral immune response, are the main force driving the battle against foreign invaders.     

昆虫天然免疫反应分子机制研究进展

昆虫体内缺乏高等脊椎动物所具有的获得性免疫系统, 只能依赖发达的天然免疫系统抵抗细菌、 真菌、 病毒等外源病原物的侵染。本文概括了昆虫天然免疫反应发生和作用的分子机制相关进展, 重点阐述了重要免疫相关因子在昆虫天然免疫反应中的功能和作用机制。昆虫天然免疫反应分为体液免疫和细胞免疫两种, 二者共同作用完成对病原物的吞噬 (phagocytosis)、 集结 (nodulation)、 包囊 (encapsulation)、 凝结 (coagulation)和黑化（melanization）等。当昆虫受到外界病原物的侵染时, 首先通过体内的模式识别蛋白（pattern recognition proteins/receptor, PRPs）识别并结合病原物表面特有的模式分子（pathogen-associated molecular pattern, PAMPs）, 继而一系列包括丝氨酸蛋白酶和丝氨酸蛋白酶抑制剂在内的级联激活反应被激活和调控, 产生抗菌肽、 黑色素等免疫效应分子, 清除或杀灭外源物。抗菌肽是一类小分子量的阳离子肽, 具有广谱抗菌活性, 针对不同类型的病原物, 抗菌肽的产生机制也不尽相同。昆虫体内存在着两种信号转导途径调节抗菌肽的产生： 一是由真菌和大部分革兰氏阳性菌激活的Toll途径; 二是由革兰氏阴性菌激活的Imd途径（immune deficiency pathway）。这两个途径通过激活不同转录因子调控不同抗菌肽基因的表达参与昆虫体内的天然免疫反应。     

Brugia malayi and Brugia pahangi: inherent difference in immune activation in the mosquitoes Armigeres subalbatus and Aedes aegypti

The inherent ability of Brugia malayi and Brugia pahangi (Nematoda) to establish successful relationships with the mosquitoes Armigeres subalbatus and Aedes aegypti Liverpool strain was evaluated. Brugia pahangi microfilariae (mff) avoided the immune response and developed normally in A. subalbatus exposed to the parasite by an infective bloodmeal, whereas nearly 85% of B. malayi were destroyed by the immune response. Because A. aegypti supports the development of both filarial worm species but destroys intrathoracically inoculated B. pahangi isolated from jird blood, blood-isolated B. malayi were inoculated into A. aegypti, and the immune response was compared with that observed against B. pahangi. The response against B. malayi was significantly more rapid and effective than the response against B. pahangi. Similar results were obtained when blood-isolated B. pahangi or B. malayi were inoculated into A. subalbatus. Microfilariae of both species were able to avoid immune destruction in A. aegypti if they were allowed to penetrate the Liverpool midgut in vitro prior to inoculation. Most B. pahangi that had first penetrated an Armigeres midgut prior to inoculation into A. subalbatus were able to avoid the immune response, but by day 3 postinoculation, less than 40% of the B. malayi, treated in the same manner, were able to escape the immune response. Genetic susceptibility of mosquitoes to infection by filarial worms and potential mechanisms of immune evasion/suppression are discussed regarding B. malayi and B. pahangi.     

Dirofilaria immitis: effect on hemolymph polypeptide synthesis in Aedes aegypti during melanotic encapsulation reactions against microfilariae

[35S]Methionine-labeled hemolymph polypeptides from adult, female Aedes aegypti Liverpool strain mosquitoes inoculated with the microfilariae of the filarial nematode Dirofilaria immitis were compared with those from saline-inoculated and uninoculated controls by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by fluorography. SDS-PAGE analysis of cell-free hemolymph collected via perfusion at 6, 12, 24, 48, 72, and 96 hr postinoculation (PI) detected the enhanced expression of an 84-kDa polypeptide. This polypeptide, expressed constitutively in the hemolymph of all three groups of mosquitoes, increased considerably in inoculated mosquitoes as time progressed as compared with uninoculated controls. Moreover, the 84-kDa polypeptide was expressed at higher levels in D. immitis-inoculated mosquitoes than in saline-inoculated controls. This stimulation of de novo biosynthesis of the 84-kDa polypeptide in inoculated mosquitoes may play a role in the immune response of mosquitoes. Since it is likely that the wound healing response in insects involves many of the same chemical processes as occur in melanotic encapsulation reactions of mosquitoes against filarial worms, the preferential expression of the 84-kDa polypeptide in saline-inoculated mosquitoes seen in this study may reflect a wound healing response. The greater increase in synthesis of this protein in D. immitis-inoculated mosquitoes may reflect production of melanotic material required for parasite destruction as well as for wound healing.     

Hemocyte monophenol oxidase activity in mosquitoes exposed to microfilariae of Dirofilaria immitis

Monophenol oxidase (MPO) activity in hemocytes collected from Aedes aegypti Liverpool strain and Aedes trivittatus intrathoracically inoculated with saline alone, inoculated with Dirofilaria immitis microfilariae (mff), or from uninoculated mosquitoes was compared using a radiometric tyrosine hydroxylation assay. Hemocyte MPO activity in mff-inoculated (= immune-activated) mosquitoes was significantly increased at 24 hr postinoculation (PI) in A. aegypti and at 6, 12, and 24 hr PI in A. trivittatus as compared with saline-inoculated controls. Baseline and immune-activated levels of hemocyte MPO activity in A. trivittatus were significantly higher compared with those seen in A. aegypti. Baseline hemocyte population levels were similar in both species, but immune activation did not elicit increases in total hemocyte populations in A. trivittatus as has been demonstrated for A. aegypti. Likewise, immune activation by the inoculation of mff did not significantly alter plasma MPO activity in A. trivittatus as compared with uninoculated or saline-inoculated mosquitoes. Plasma MPO activity in A. aegypti, however, appears to constitute a major component of the immune response. The importance of phenol oxidase(s) in the immune response of mosquitoes against mff and the relationship of observed differences in MPO activity to differences in immunological capability between A. aegypti and A. trivittatus are assessed.     

Aedes aegypti genomics

The mosquito, Aedes aegypti, is the primary, worldwide arthropod vector for the yellow fever and dengue viruses. As it is also one of the most tractable mosquito species for laboratory studies, it has been and remains one of the most intensively studied arthropod species. This has resulted in the development of detailed genetic and physical maps for Ae. aegypti and considerable insight into its genome organization. The research community is well-advanced in developing important molecular tools that will facilitate a whole genome sequencing effort. This includes generation of BAC clone end sequences, physical mapping of selected BAC clones and generation of EST sequences. Whole genome sequence information for Ae. aegypti will provide important insight into mosquito chromosome evolution and allow for the identification of genes and gene function. These functions may be common to all mosquitoes or perhaps unique to individual species, possibly specific to host-seeking and blood-feeding behaviors, as well as the innate immune response to pathogens encountered during blood-feeding. This information will be invaluable to the global effort to develop novel strategies for preventing arthropod-borne disease transmission.     

Defense reactions of mosquitoes to filarial worms: effect of host age on the immune response to Dirofilaria immitis microfilariae

The melanization response of Aedes aegypti black-eyed Liverpool strain (LVP) and Aedes trivittatus against intrathoracically inoculated Dirofilaria immitis microfilariae (mff) was assessed in mosquitoes less than 1, 14, 21, and 28 days after adult ecdysis. There was a significant decrease in the melanization response of A. aegypti 14 days of age and older at 1, 3, and 5 days postinoculation (PI) compared to less than 1-day-old mosquitoes. The response also was reduced significantly in 14- to 28-day-old A. trivittatus on days 1 and 3 PI. Although essentially 100% of recovered mff were melanized by day 5 PI in A. trivittatus, the amount of melanin deposited was much less than that seen in 0-day-old mosquitoes. Potential mechanisms responsible for a reduced immune competence in older mosquitoes and the possible relationship to vector potential are discussed.     

Induction of salivation in biting midges and mosquitoes, and demonstration of virus in the saliva of infected insects

Culicoides biting midges and Aedes aegypti (Linnaeus) mosquitoes were induced to salivate by the topical application of pilocarpine, neostigmine, malathion and dimethoate; of these, malathion was the most effective. Drops of saliva produced by virus-infected midges and mosquitoes were shown to contain virus. The method could be used to demonstrate transmission in insects infected with a variety of pathogens.     

No maternal effects after stimulation of the melanization response in the yellow fever mosquito Aedes aegypti

The costs and benefits of activating the immune system can reach across generations. Thus, in vertebrates and in several invertebrates, stimulating the immune system of a female can enhance immunity of her offspring or decrease offspring fitness. We evaluated the potential maternally transmitted costs and benefits of the melanization response, an innate immune response of insects that helps to protect mosquitoes from malaria parasites. We manipulated the maternal melanization response of the yellow fever mosquito Aedes aegypti by inoculating female mosquitoes with negatively charged sephadex beads or with immunologically inert glass beads; a control group was not inoculated. In the next generation, we assayed the melanization response and measured three other life-history traits: survival up to emergence, the age at emergence, and body size (estimated as wing length). We found no evidence of fitness costs or benefits for the offspring. A retrospective power analysis found that our experiment would have detected an effect size that is three times smaller than the maternal immune priming effects that have been reported in the literature. We did find a strong correlation between offspring wing length and melanization response. Overall, our findings indicate that trans-generational immune priming in invertebrates cannot be generalized, and that it may depend on the species, the immune challenge, and the environmental conditions.     

Effect of Cecropin B and a Synthetic Analogue on Propagation of Fish Viruses In Vitro

Abstract Cecropins and other natural antimicrobial peptides are widely distributed in animals from insects to mammals. These proteins have been shown to be major constituents of the innate immune systems of animals for nonspecific defense of the host against various bacteria and parasites. Therefore, exploitation of this natural innate defense system may lead to the development of effective methods for protecting fish from invasion by microbial pathogens. Recently, we have demonstrated that the introduction of cecropin transgenes into Japanese medaka (Oryzias latipes) conferred resistance to infection by fish bacterial pathogens. Aside from a few reports documenting the antiviral effect of antimicrobial peptides including cecropins against mammalian viruses, there is no evidence for the effect of these peptides against fish viruses. In this article we present results of in vitro characterization of native cecropin B and a synthetic analogue, CF17, against several important fish viral pathogens—namely, infectious hematopoietic necrosis virus (IHNV), viral hemorrhagic septicemia virus (VHSV), snakehead rhabdovirus (SHRV), and infectious pancreatic necrosis virus (IPNV). Upon coincubation of these peptides and viruses, the viral titers yielded in fish cells were reduced from several fold to 104-fold. Direct disruption of the viral envelope and disintegration of the viral capsids may be involved in the inhibition of viral replication by the peptides. Results of our studies demonstrate the potential of manipulating antimicrobial peptide genes by transgenesis to combat viral infection in fish.     

Parasite-induced suppression of the immune response in Aedes aegypti by Brugia pahangi

The melanization response against intrathoracically inoculated Brugia pahangi and Dirofilaria immitis microfilariae (mff) isolated from vertebrate host blood was evaluated in both uninfected Aedes aegypti black-eyed Liverpool strain and in mosquitoes harboring a developing B. pahangi infection. The immune response against inoculated mff of either species was significantly reduced by 28-47% in infected as compared with uninfected mosquitoes. Attempts to passively transfer this suppression factor(s) by inoculating naive mosquitoes with 0.1-0.2 microliter of hemolymph from B. pahangi-infected mosquitoes produced equivocal results. The role this parasite-induced immune suppression might play in aiding parasite survival in compatible vectors is discussed.     

Induction of a peptide with activity against a broad spectrum of pathogens in the Aedes aegypti salivary gland, following Infection with Dengue Virus

The ultimate stage of the transmission of Dengue Virus (DENV) to man is strongly dependent on crosstalk between the virus and the immune system of its vector Aedes aegypti (Ae. aegypti). Infection of the mosquito's salivary glands by DENV is the final step prior to viral transmission. Therefore, in the present study, we have determined the modulatory effects of DENV infection on the immune response in this organ by carrying out a functional genomic analysis of uninfected salivary glands and salivary glands of female Ae. aegypti mosquitoes infected with DENV. We have shown that DENV infection of salivary glands strongly up-regulates the expression of genes that encode proteins involved in the vector's innate immune response, including the immune deficiency (IMD) and Toll signalling pathways, and that it induces the expression of the gene encoding a putative anti-bacterial, cecropin-like, peptide (AAEL000598). Both the chemically synthesized non-cleaved, signal peptide-containing gene product of AAEL000598, and the cleaved, mature form, were found to exert, in addition to antibacterial activity, anti-DENV and anti-Chikungunya viral activity. However, in contrast to the mature form, the immature cecropin peptide was far more effective against Chikungunya virus (CHIKV) and, furthermore, had strong anti-parasite activity as shown by its ability to kill Leishmania spp. Results from circular dichroism analysis showed that the immature form more readily adopts a helical conformation which would help it to cause membrane permeabilization, thus permitting its transfer across hydrophobic cell surfaces, which may explain the difference in the anti-pathogenic activity between the two forms. The present study underscores not only the importance of DENV-induced cecropin in the innate immune response of Ae. aegypti, but also emphasizes the broad-spectrum anti-pathogenic activity of the immature, signal peptide-containing form of this peptide.     

Self-harm caused by an insect's innate immunity

It has been a long-held assumption that the innate immune system of insects causes self-harm when used to combat an immune insult. We show empirically that this assumption is correct. Invertebrate innate immunity relies heavily on effector systems which, on activation, produce cytotoxins that kill pathogens. Reliance on these robust, fast-acting, generic killing mechanisms ensures a potent and rapid response to pathogen invasion, but has the potential disadvantage of causing self-damage. We show that the innate immune response against an immune insult produces measurable phenotypic and functional damage to self-tissue in the beetle Tenebrio molitor. This type of self-harm (autoreactivity) and the life-history implications that arise from it are important to understand evolutionary phenomena such as the dynamics between hosts and parasites as well as the nature of immune system costs.     

The Aedes aegypti toll pathway controls dengue virus infection

Aedes aegypti, the mosquito vector of dengue viruses, utilizes its innate immune system to ward off a variety of pathogens, some of which can cause disease in humans. To date, the features of insects' innate immune defenses against viruses have mainly been studied in the fruit fly Drosophila melanogaster, which appears to utilize different immune pathways against different types of viruses, in addition to an RNA interference-based defense system. We have used the recently released whole-genome sequence of the Ae. aegypti mosquito, in combination with high-throughput gene expression and RNA interference (RNAi)-based reverse genetic analyses, to characterize its response to dengue virus infection in different body compartments. We have further addressed the impact of the mosquito's endogenous microbial flora on virus infection. Our findings indicate a significant role for the Toll pathway in regulating resistance to dengue virus, as indicated by an infection-responsive regulation and functional assessment of several Toll pathway-associated genes. We have also shown that the mosquito's natural microbiota play a role in modulating the dengue virus infection, possibly through basal-level stimulation of the Toll immune pathway.