Research progress on the diversity and application of mosquito intestinal flora

Biao Liu, Xun Kang, Yue-Xin Wang, Xing-Yong Wu, Hui-Min Fu, Le Kang,Qian-Feng Xia✉

1. Laboratory of Tropical Biomedicine and Biotechnology, Hainan Medical University, Haikou 571199, China

2. Key Laboratory of Tropical Translalional Medicine of Ministry of Education and School of Tropical Medicine and Laboratory Medicine, Hainan medical University, Haikou 571199, China

3. State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China

ABSTRACT Mosquitoes are an important medical insect and play a very important role in the process of biologically vector infectious diseases. A large number of bacterial communities are colonized in their intestines. In the process of interaction and symbiosis with mosquitoes, the bacterial community is in many ways. The life activities of mosquitoes have a significant impact. This article reviews the population structure of the mosquito intestinal flora, influencing factors, and the regulation of mosquito vector efficacy by the flora.

Keywords:Mosquitoes Intestinal flora Diversity Media effectiveness✉Corresponding author: XIA Qian‑feng, Professor, Tutor of Ph.D. candidates.E‑mail: xiaqianfeng@hainmu. edu. cn;KANG Le,E‑mail:lkang@ioz.ac.cn.

Mosquitoes are closely related to human life and are very important medical vectors. By biting people and vertebrates,Dengue Virus, Zika Virus, Plasmodium and other pathogens that can be transmitted [1-5] have posed huge challenges to public health prevention and control in the world. As the mosquito's first important defense line against pathogenic microorganisms, the gut colonizes a large number of microflora, including plankton (aquatic algae, etc), bacteria and fungi, etc. At present, bacteria have attracted extensive attention in the research on the intestinal microorganisms of mosquitoes [6].Since the launch of the Human Microbiome Project (HMP, 2007 NIH) in 2007, more and more studies have shown that the microbiome is closely related to the life activities of the host in many aspects in the process of interaction and symbiosis with the host [7, 8]. In this paper, the diversity and influencing factors of the intestinal flora of mosquitoes, as well as the influence of intestinal flora on mosquito life activities and vector efficacy were reviewed.

1. Diversity of intestinal flora of mosquitoes

Before larvae emerge into adults, mosquitoes mainly breed in the environment of stagnant water. Different mosquito species have different requirements for water quality, and the environment of adults is relatively complex and changeable compared with that of larvae because they can fly [9]. From the source of food for analysis,larvae in single‑celled organisms, organic detritus and small invertebrates, males are usually feed on the pollen nectary, while adult females with smoking vertebrate blood as the main source of food [10, 11], and it also provides the necessary nutrition to lay eggs,and the relative special food chain as well as pathogenic organisms in the communication between the host provides the necessary way to [12]. Most of the intestinal flora colonized by mosquitoes are obtained from the environment mainly through diet, and only a few are obtained directly through vertical transmission [13, 14]. This view is supported by a number of research data. First, experimental studies have found that there is no extracellular flora in the intestinal tract when mosquito larvae hatch. Secondly, most of the mosquito intestinal flora identified in larvae were highly overlapping with those found in their aquatic habitats. Third, mosquitoes have highly variable intestinal communities [6, 15-17]. With the rapid development of sequencing technology, the study on the structure of mosquito intestinal flora has changed from in vitro bacterial culture to in vitro bacterial culture combined with metagenomic sequencing technology [18], which greatly promoted the investigation and study of bacteria that could not be cultured in vitro. At present, besides rare is difficult to identify the sequence of mosquito has identified the intestinal bacterial community diversity compared with vertebrate,mosquitoes in the intestinal bacterial community diversity are relatively low (about 200) [12], but from the mosquito's gut bacteria community diversity and other intestinal flora of metabolic insects from environment, compared to the intestinal flora composition is still very considerable [13]. Different mosquito species, habitat preferences different, urvive Aaedes larvae in homes around the container water (cylinder, cans, POTS, bamboo tube, tree holes, tires,etc) is given priority to, Armigeres subalbatus larvae are survive indoors, barns, toilet water endogenous habitation, Anopheles larvae survive with shade, water quality of cool and refreshing rice fields, marshes, flooding, such as environment is given priority to, whereas Culex survive in housing near sewage or water tanks,adult because of its flight, and need to find suitable spawning environment, its unhealthy with wider range of environments [10, 11,19]. Studies have shown that in the larva and adult stages, most of the identified bacteria in the gut are mainly Gram‑negative aerobes or facultative anaerobes, mainly belonging to four dominant phyla,namely Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria[6, 15-17, 20-23]. Common colonization strains include Elizabeth and Chryseobacterium of Bacteroidetes, Pseudomonadaceae, Serratia,Wolbachia, Enterobacter and Acinetobacter of Proteobacteria,Asaia and Pantoea were also commonly found in the intestines of Aedes and Anopheles mosquitoes [16, 24, 25]. Study found that the same mosquito larvae and adult on the basis of different habitats and the influence of growth and development, in most cases, the larvae of bacteria species diversity is always higher than adult [6,15, 17, 22], and laboratory breeding mosquito species compared with the same mosquito species of field acquisition, the lower intestinal flora diversity [15, 16, 20], and field collection of mosquito, mosquito gut flora diversity than male [24]. Found in the study of multiple sampling points, the individuals of the same or different species in the same place and the same of the intestinal flora from the acquisition date than individuals from different place and the same acquisition date intestinal flora more similar [6, 16, 22], but there is no evidence to prove that in the same sampling area and the same or different kind of mosquito larvae and adult gut is a particular [26]"core" flora. Most research results happen to coincide to diversity within and between different mosquito species of mosquito bacteria by different collection sites have bigger difference, the same place where the mosquito gut can have similar bacterial flora structure [6,16, 17, 21, 22, 27-29], and the mosquito of intestinal flora was obtained from the environment and other insects and vertebrates relevant research conclusions similar [30], this a series of research shows that mosquitoes in the formation of the habitat structure of intestinal flora plays a dominant role. In a particular region, the influence of intestinal flora secondary factors include the PH of the insect eating foods, carbohydrate content, metabolic pathway, etc all can affect the structure of intestinal flora [20, 31, 32], intestinal flora also changes with age and changing, perhaps this is the intestinal flora with mosquitoes competition sugar source related [33, 34]. However,Plasmodium vivax can induce early inhibition of intestinal flora by regulating iron metabolism in the intestine of Anopheles mosquitoes[34], thus causing changes in intestinal flora.

2. The influence of intestinal flora on the growth and development of mosquitoes

Ingut flora plays a very important role in the growth and development of mosquitoes [7, 8], providing necessary nutrients to mosquitoes through different ways [35, 36]. Earlier studies have found that when the number of bacteria in aquatic environment to reduce, the mosquitoes in the larval stage show higher mortality rates or delay to the growth of the pupal stage [22], in Aedes aegypti since eggs cultivation, through constructing sterile larvae, innocuity in comparison with the control group of normal breeding, also found that the experimental group do not grow, and a instars death after a few days, in artificially added after normal bacteria or Saccharomyces, can restore its normal growth [15, 37]. Interestingly,the researchers found that only feeding live bacteria supported the growth and development of Aedes aegypti, as has been demonstrated in several other mosquito species [15, 17, 22], and gut bacteria were also found to be critical for molting larvae [38]. In a recent study s hows that under the cultivation of the habitat no flora, given normal nutrients, still can bring from eggs develop into adult mosquitoes Aedes aegypti and continue to develop to the next generation, sterile insect development is normal, only exist in the development of the time delay [39], on the grant of anopheles improve the abundance of bacteria in food can accelerate the growth and development of larval[31], the evidence of intestinal flora of secondary metabolites may be adjust the influence factors of mosquito growth is very important.Studies have also found that Bacillus thuringiensis subspecies can inhibit the growth and development of Chironomids [40], while the intestinal bacteria of Aedes aegypti can affect its digestion of blood meal, the mechanism of which is thought to be that Serrella and Enterobacter can promote the absorption of nutrients by mosquitoes after blood feeding through the production of hemolytic enzyme[41]. So far, most of the specific mechanisms of inte stinal flora influencing mosquito growth and development have not been studied and identified, but these series of apparent phenomena still indicate that intestinal flora and secondary products play an indispensable role in every stage of mosquito life.

3. Effects of intestinal flora on immune regulation in mosquitoes

Vector efficiency refers to the ability of mosquitoes to replicate,proliferate and transmit pathogens to other hosts after they infect pathogens [42]. Intestinal flora maintains a parasitic or symbiotic relationship with mosquito hosts for a long time. During their own life activities, the flora and its secondary products influence the vector efficiency of mosquitoes in multiple ways, and play an important role in the development and function of the mosquito immune system [43, 44]. Studies have shown that intestinal flora by infected mosquitoes can activate intestinal immune gene expression[33], and Anopheles infected after different of Serratia species, its efficiency of the malaria parasite infection also change [45], and Serratia bacteria can also promote the Dengue Virus, Village Card Virus, Chikungunya Virus of mosquito infection [46-48], the present study considered the mechanism for Serratia bacteria through the secretion of peptides in the combination of Aedes aegypti value‑added resistant protein on the surface of the bowel, thereby promote mosquito susceptible of Dengue Virus, and aromatic Serratiat through P40 secreted protein, in addition, it can combine with pore proteins on mitochondria of midgut cells of mosquitoes to inhibit mosquito immunity and promote Chikungunya Virus infection [46,47].

A variety of antimicrobial peptide genes in intestinal epithelial cells of Aedes aegypti infected by Proteus can be activated and expressed to enhance the mosquito immunity to the virus [49]. More interesting finding is Chromobacterium can not only inhibit the growth of a variety of bacteria in the intestines [50], there is a wide range of antiviral activity, it not only can significantly reduce the Anopheles Gambiae of plasmodium infection, also can reduce the Dengue Virus infection of Aedes aegypti, but the specific mechanism of action of research at present there are not many [51]. After eating blood containing antibiotics, Anopheles Gambiae has reduced its immunity to malaria parasites [36]. Moreira team first reported theo buck Wolbachia by infected Aedes aegypti, can reduce the dengue virus and Chikungunya Virus infection Aedes aegypti [52], the ability of the recent reports also indicate that theo buck's physical ability significantly inhibit village Zika Virus infection Aedes a egypti[53], the reported theo buck Wolbachia in Aedes aegypti immune regulation is, the way of a wall, buck Wolbachia by infected Aedes aegypti, activation of mosquito ROS‑Tall, in order to enhance the immune power of the Aedes aegypti, reduce the susceptibility of pathogens and parasites Aedes aegypti [54]. By infecting Anopheles mosquitoes, the bacteria can activate the immune system of Anopheles mosquitoes and significantly inhibit the formation of oocysts of Plasmodium parasites, thus reducing the transmission of Plasmodium [55]. Theo buck Wolbachia for maternal genetic [56], in the insect reproductive process of Cytoplasmic incompatibility is the wall Wolbachia to buck the basis for the control of insect populations based on Cytoplasmic incompatibility mosquito birth control technology has been more field experiment [57], and proved that the method can effectively control the Aedes albopictus population [58].The other team infected the bacteria Bacillus Sphaericus through the intestines of Anopheles mosquitoes, and used the MTX and Bin virulence protein synthesized by the bacteria to effectively kill Anopheles trumbus, thus achieving the control of Anopheles population and achieving the purpose of biological mosquito control and prevention of infectious diseases [59]. In biological mosquito another widely attention is using Xenorhabdus and Photorhabdus different species of the genus to implement biological mosquito[60, 61], has now found 24 strains Xenorhabdus and Photorhabdus 5 strains have kill mosquitoes features, and the ways to kill mosquitoes to consider is that these strains by infected mosquitoes, bacterial genes encoding low molecular weight compounds and protein,secondary toxin has active metabolites of pesticides, the mosquito species ill injury and even death, so as to achieve mosquito, cut off the pathogen transmission way, A number of studies have proved that they are effective against mosquitoes and friendly to the environment and humans [ 62-68]. The results of this series of studies provide a research basis for the development of more compounds and biological agents for mosquito control in the future.

4. Prospection

The spread of mosquitoes to infectious diseases is still one of the huge challenges facing global public health security, and mosquito control is still the best choice to deal with. However, the traditional chemical anti‑mosquitoes, which are not friendly to the living environment and rapidly produce drug‑resistant mosquitoes, make it more urgent to find new mosquito control methods. In recent years,through the display of in‑depth research results on the interaction between mosquitoes, intestinal flora and pathogens, new ideas have been provided for biological anti‑mosquito and prevention of vecto‑borne diseases. With the further development of high‑throughput sequencing technology, bioinformatics technology, metagenomics and other related technologies, it has greatly promoted the related research of mosquitoes and intestinal flora, and demonstrated that the intestinal flora plays a role in mosquito immune regulation, the powerful regulation of biological media, population control, growth and development etc, reveals the mechanism of the influence of intestinal flora on mosquitoes from many aspects. In order to further clarify the transmission factors of mosquito‑borne diseases and the application of biological mosquito control and prevention and control of vector‑borne diseases, new directions have been opened up.