Isolation and identification of Asaia sp. in Anopheles spp. mosquitoes collected from Iranian malaria settings: steps toward applying paratransgenic tools against malaria

Abstract

BackgroundIn recent years, the genus Asaia (Rhodospirillales: Acetobacteraceae) has been isolated from different Anopheles species and presented as a promising tool to combat malaria. This bacterium has unique features such as presence in different organs of mosquitoes (midgut, salivary glands and reproductive organs) of female and male mosquitoes and vertical and horizontal transmission. These specifications lead to the possibility of introducing Asaia as a robust candidate for malaria vector control via paratransgenesis technology. Several studies have been performed on the microbiota of Anopheles mosquitoes (Diptera: Culicidae) in Iran and the Middle East to find a suitable candidate for controlling the malaria based on paratransgenesis approaches. The present study is the first report of isolation, biochemical and molecular characterization of the genus Asaia within five different Anopheles species which originated from different zoogeographical zones in the south, east, and north of Iran.MethodsMosquitoes originated from field-collected and laboratory-reared colonies of five Anopheles spp. Adult mosquitoes were anesthetized; their midguts were isolated by dissection, followed by grinding the midgut contents which were then cultured in enrichment broth media and later in CaCO3 agar plates separately. Morphological, biochemical and physiological characterization were carried out after the appearance of colonies. For molecular confirmation, selected colonies were cultured, their DNAs were extracted and PCR was performed on the 16S ribosomal RNA gene using specific newly designed primers.ResultsMorphological, biochemical, physiological and molecular results indicated that all isolates are members of the genus Asaia.ConclusionsContrary to previous opinions, our findings show that Asaia bacteria are present in both insectary-reared colonies and field-collected mosquitoes and can be isolated by simple and specific methods. Furthermore, with respect to the fact that we isolated Asaia within the different Anopheles specimens from distinct climatic and zoogeographical regions, it is promising and may be concluded that species of this genus can tolerate the complicated environmental conditions of the vector-borne diseases endemic regions. Therefore, it can be considered as a promising target in paratransgenesis and vector control programs. However, we suggest that introducing the new technologies such as next generation sequencing and robust in silico approaches may pave the way to find a unique biomarker for rapid and reliable differentiation of the Asaia species.

Leucine-Rich Immune Factor APL1 Is Associated With Specific Modulation of Enteric Microbiome Taxa in the Asian Malaria Mosquito Anopheles stephensi

“…The Anopheles ortholog of Down syndrome cell adhesion molecule gene, AgDscam, displays differential transcript splicing and protein binding to several bacteria tested, although a role as an enteric barrier factor policing the microbiome is not known (Dong et al, 2006). Bacteria of the genus Asaia are found in the enteric microbiome of wild Anopheles and confer host benefit, but host mechanisms of their maintenance as commensals are not known (Chouaia et al, 2012;Rami et al, 2018). In Aedes mosquitoes, C-type lectins coat the surface of enteric bacteria and protect them from attack by antimicrobial peptides (Pang et al, 2016).…”

Mosquito-Borne Diseases Emergence/Resurgence and How to Effectively Control It Biologically

“…The simplest approach to this is paratransgenesis, consisting of producing bacterial strains that are able to both live in the midgut of various mosquito species and spread rapidly among wild mosquito populations [164]. Several studies have been performed on the identification and use of competent microorganisms to combat vector-borne diseases [165]. The genus Asaia, first discovered in plant nectar, is an excellent candidate [166]; it is localized in many organs of mosquitoes, and can disperse inside the mosquito body through the haemolymph [165,167].…”

“…Several studies have been performed on the identification and use of competent microorganisms to combat vector-borne diseases [165]. The genus Asaia, first discovered in plant nectar, is an excellent candidate [166]; it is localized in many organs of mosquitoes, and can disperse inside the mosquito body through the haemolymph [165,167]. Its distribution in the mosquito population is made possible through several mechanisms (co-feeding, sexual mating, paternal, maternal, and horizontal transmission) [168][169][170].…”

“…Asaia bacteria may be genetically modified in order to be recolonised in a new host, resulting in spread within wild populations [166]. Recently, it was isolated and characterised from several Anopheles species, which would be beneficial if applied toward achieving paratransgenesis against malaria [165]. Advanced studies recently showed that Asaia may activate the mosquito's immune system, leading to a reduction in the development of malaria parasites [171].…”

Chimeric symbionts expressing a Wolbachia protein stimulate mosquito immunity and inhibit filarial parasite development

“…and Wolbachia pipientis. Representatives of the genus Asaia have been detected in different mosquito species; more in general, they have been observed in several insects 2,3 . Asaia spp.…”