https://orcid.org/0000-0001-6620-8603
Figures
Abstract
The Aedes mosquitoes are responsible for the transmission of many severe diseases. Novel integrated vector management techniques like alginate hydrogel beads and appealing toxic sugar bait have strengthened control efforts. These techniques help to control mosquitoes by taking advantage of their attraction to sugar. Different types of sugar that mosquitoes ingest during feeding can affect the makeup of the microbiome in the midgut. The mosquito midgut microbiome maintains immune priming and baseline immune activity. Therefore, the current focus of this study is on utilizing microbial communities for vector control measures with a particular emphasis on how they consume various forms of sugars. Both wild and lab strains of Ae. aegypti and Ae. albopictus mosquito samples were reared, and attractive targeted sugar baits (ATSBs) infused with Chrysanthemum, mango, mix and control solutions. Then, the impact of bacterial communities was assessed using 16S rRNA gene sequences. According to our findings, the majority of the bacterial species in mango and mixed treatments belonged to the Enterobacteriaceae family. A total of 24 different bacterial species were found in Aedes mosquitoes that fed on mango ATSBs. The isolates were found to be members of three phyla from Actinobacteria (4.16%), Firmicutes (54.17%), and Proteobacteria (41.67%). Data reveals that different species, strains, and diets affect the midgut bacterial diversity in the mosquitoes. In addition to strengthening our knowledge concerning the way this bacterium shapes the microbial community, a thorough investigation of the prevalence of the midgut bacterial community is essential for alerting present and future mosquito and disease control initiatives.
Citation: Sambanthan R, Abu Kassim NF, Abuelmaali SA, Kamil WMWA, Sabar S, Zarkasi KZ, et al. (2026) Impact of sugar-based baits on midgut microbiome composition in Aedes mosquitoes: Implications for vector control. PLoS One 21(3): e0329341. https://doi.org/10.1371/journal.pone.0329341
Editor: Yara M. Traub-Csekö, Instituto Oswaldo Cruz, BRAZIL
Received: July 15, 2025; Accepted: January 30, 2026; Published: March 12, 2026
Copyright: © 2026 Sambanthan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: Fundamental Research Grant Scheme (FRGS) with Reference No: FRGS/1/2022/STG03/USM/02/8. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors declared that no competing interests exist.
Introduction
Vector control is a critical component of arbovirus prevention and, in the absence of broadly effective antivirals or vaccines for many Aedes-borne infections, remains the primary strategy recommended by global health agencies to mitigate outbreaks [1–3]. Among emerging tools, attractive toxic sugar baits (ATSBs) are particularly promising because they exploit the natural sugar-feeding behaviour of mosquitoes by combining plant-derived attractants (such as sucrose, nectar, and fruit volatiles) with orally active insecticides to induce lethal ingestion during sugar meals [4,5].
Field and semi-field trials have shown that ATSBs can substantially reduce Aedes mosquito populations, underscoring their potential as an important addition to integrated vector management. Because ATSBs act through ingestion and directly modify the nutritional and chemical environment of the midgut, they also have the capacity to alter the mosquito gut microbiome, a community increasingly recognised as a key determinant of mosquito physiology and vector competence [6–8]. The term “microbiome” refers to the grouping of all microorganisms that naturally reside on or within insect bodies, including bacteria, fungi, viruses, and their genes. The microbiome acts as a key interface between the insect body and the environment. It will affect the current condition of the body by altering the response to environmental changes. Certain microorganisms function as a buffer, reducing the toxicity of environmental contaminants, and vice versa [5,9,10]. The salivary glands, crop, gut, ovaries, Malpighian tubules, and hemocoel are among the many organs that mosquito microbial symbionts reside in [5,9,11]. The developmental process and transmission of viruses and parasites rely on these organs. The gut is the primary site of pathogen exposure because of the blood meal storage and metabolization processes that occur in the mosquito’s midgut.
The gut microbiota of mosquitoes is predominantly composed of Gram-negative bacteria [12]. It was primarily composed of bacteria from the families Oxalobacteraceae, Enterobacteriaceae, Comamonadaceae and the genera Pseudomonas, Acinetobacter, Aeromonas, and Stenotrophomonas [13]. Reports indicate that midgut bacteria significantly influence disease transmission potential, as they are associated with host-pathogen interactions and, ultimately, vector competence [5,14]. These results point to a unique disease control strategy by introducing particular bacterial strains with innate or designed anti-pathogen activity into the mosquito gut [15,16].
The identification and classification of bacteria and archaea communities is frequently accomplished by the use of 16S ribosomal RNA (rRNA) sequencing [17]. The small 30S subunit of a prokaryotic ribosome is made up of the bacterial 16S rRNA, with about 1500 bp length [18]. Generally, this gene consists of nine hypervariable regions interspersed with conserved sections. Several bacterial species share a high degree of conservation in the 16S rRNA gene’s conserved regions. The nine hypervariable regions (V1–V9) are informative for phylogenetic analysis and species identification because they show significant sequence diversity among various bacterial species and contain important information about genetic differences between bacterial species [19].
The composition of the gut microbiota in mosquitoes is very dynamic and strongly influenced by dietary inputs, especially sugar feeding. The main energy source for both male and female mosquitoes is nectar or sugars derived from plants. Sugar meals are known to change the pH, redox conditions, and availability of nutrients in the midgut, which in turn shapes the structure of microbial communities [20]. The mosquito midgut represents the most important site for host-pathogen interaction, consisting of a digestive tract that is responsible for their food digestion. The communities of microbiota play critical roles in their biological processes, such as immune response function, sexual reproduction, digestion, development, and refractoriness of pathogens [21]. Sugar feeding can alter the variety and number of bacteria in the mosquito’s midgut, affecting survival, metabolism, immunological function, and vector competence. Dietary modifications, for example, have been demonstrated to influence midgut microbial populations as well as antiviral or antiparasitic responses, impacting infection susceptibility and transmission potential. The influence of natural sugar sources on mosquito gut microbiota composition and function is not well understood, creating a significant gap for sugar-based control measures [22].
This knowledge gap is particularly relevant in the context of Attractive Toxic Sugar Baits (ATSB), an emerging vector control strategy that exploits the natural sugar-feeding behavior of mosquitoes. ATSB consist of sugar-based attractants combined with oral toxins and are designed to reduce mosquito populations by inducing lethal ingestion during sugar feeding. Plant-based sugar sources are often incorporated into ATSB formulations to enhance attractiveness and feeding rates. However, the interaction between ATSB-associated sugar sources and the mosquito gut microbiota is poorly understood, and changes in microbial composition could potentially influence feeding behavior, toxin susceptibility, and overall control efficacy [8].
Mango (Mangifera indica), Chrysanthemum spp., and a sucrose solution were chosen for this investigation as representative natural and artificial sugar sources that are pertinent to mosquito sugar feeding and have been utilized in the creation of ATSB formulations [5,9]. These substrates are good models for studying diet-driven changes in the mosquito gut microbiota because of their differences in chemical composition, microbial loads, and secondary metabolite profiles. In order to provide fundamental insights into mosquito biology and the optimization of sugar-based vector control techniques, this exploratory work sought to expand on the current understanding of midgut microbiome diversity in mosquitoes after eating on various sugar sources.
Materials and methods
Sample collection and rearing
The eggs of laboratory strains of Ae. aegypti and Ae. albopictus were obtained from the Vector Control Research Unit (VCRU), School of Biological Sciences (SBS), University of Science Malaysia. The rearing process was carried out at the Insectary Building G025A-Medical Entomology Laboratory, SBS. The rearing process was conducted under the condition where the temperature was maintained at 27 °C (± 2 °C). Meanwhile, the relative humidity and photoperiod were maintained at 75% (± 3%) and 12:12 h (L:D), respectively. The eggs were hatched in dechlorinated water, and the newly emerged larvae were transferred to metallic trays filled with dechlorinated water (depth = 2 cm, diameter = 12 cm). The larvae were fed daily for two days with a 2:1:1:1 ratio of fine powder formed by a mixture of cat food, beef, liver, yeast, and milk powder [23]. Then, the pupae were placed in a 250 mL capacity plastic cup that was kept in mosquito rearing cages measuring 30 cm × 30 cm × 30 cm. The newly emerged adult was provided with a 10% sucrose solution as a food source. The eggs of wild strain Ae. aegypti and Ae. albopictus were collected around the University of Science Malaysia and were reared in Insectary Building G025A-Medical Entomology Laboratory, SBS, under the same condition of laboratory strain. This study was approved by USM/IACUC/2022/ (137) (1223).
ATSBs preparation
The Mango (Waterlily Thai Mango) and chrysanthemum were purchased from Lotus Sungai Dua, Penang, Malaysia. The baits were prepared in a sterile condition in the laboratory. Mango extract was obtained by peeling off the skin of the mango and cutting it into small pieces. Then, distilled water was added to the mango pieces with a dilution ratio of 2:1 (distilled water: mango). It was then blended, and the solution was filtered using filter paper. Extracted mango solution was used to make 30% v/v of mango (MG) ATSBs. Thus, 120 mL of mango solution and 280 mL of distilled water were mixed. Then, to produce the ATSBs, 5 g of sodium alginate powder (Sigma-Aldrich, St. Louis, MO, USA) was weighed and blended with the mixture until a homogenous suspension formed. It was then sonicated in an ultrasonic bath (BactoSonic®) at 40 °C for 45 minutes. After 45 minutes, the emulsion was cooled to room temperature. Formation of the spherical ATSBs was carried out by dropping the emulsion into 0.2 M calcium chloride dihydrate (Sigma-Aldrich, St. Louis, MO, USA) CaCl2 solution at a rate of 300 rpm at 30 °C for the cross-linking process. Finally, the ATSBs were filtered and washed using distilled water three times to ensure that the excess chloride ion was removed from them. For 30% v/v Chrysanthemum (FL) ATSBs, the petals were detached from the flowers and weighed. Then, it was blended by using distilled water to the ratio of 2:1 (distilled water: Chrysantemum petals) to obtain the Chrysanthemum solution. Sucrose ATSBs were the control ATSBs for this study, produced by using 10% of sucrose solution. The spherical shape formation process of ATSBs for 30% v/v Chrysanthemum, 30% v/v mix of mango & Chrysanthemum (MX), and sucrose ATSBs were done by following the steps that were used for 30% v/v of mango ATSBs production.
The ATSBs are used as the medium to deliver plant extracts. It is made of sodium alginate, which is a naturally occurring anionic polysaccharide that can be taken from marine brown algae like Laminaria hyperborea, Laminaria digitate, Laminaria japonica, Ascophyllum nodosum, and Macrocystis pyrifera [24]. Usage of alginate hydrogel beads is highly recommended due to their biodegradable and non-toxic properties to the environment [25]. Additional advantageous features are high efficiency with low maintenance, and it is cheap to produce [26,27].
Sample preparation
A total of 15 male mosquitoes (three biological repeats) from the laboratory strain Ae. aegypti were placed in different cages and fed with 30% w/v of mango, chrysanthemum, mix, and control ATSBs in each cage, respectively. The experiment was repeated for both male and female laboratories and wild-strain Ae. aegypti and Ae. albopictus. The ATSBs were replaced every 24 hours because they were not treated with preservatives. Due to their important roles as arbovirus vectors and their unique ecological and behavioral traits, Ae. aegypti and Ae. albopictus were chosen. Incorporating both wild and laboratory-colonized strains enables evaluation of variations resulting from laboratory adaptation while assuring that results are applicable to natural mosquito populations. The abbreviation ALW indicates wild strain Ae. albopictus; AGW indicates wild strain Ae. aegypti; ALL refers to laboratory strain Ae. albopictus and AGL refer to laboratory strain Ae. aegypti.
After three days, mosquitoes were collected using an aspirator and killed via cold shock; they were then transferred to sterile petri dishes for dissection. To ensure sterility, the dissection area was sanitized with 70% ethanol. Using a dissecting microscope, the midguts of mosquitoes were carefully removed. For each species and sugar bait variant, five midguts from each replicate group and the control group were collected into 1.5 mL microcentrifuge tubes.
Isolation of microbial colonies from agar plate cultures
The collected midguts were sterilized with 300 µL of 70% ethanol for two minutes and rinsed three times with sterile distilled water. After removing the water, 100 µL of phosphate-buffered saline (PBS) was added to the tubes. The midguts were homogenized using a sterilized micropestle, and the homogenates were serially diluted in PBS to sevenfold dilutions. The samples were streaked onto sterile nutrient agar media and incubated at 37 °C for 24–48 hours under aseptic conditions. Plates without visible colonies were re-incubated for 16 hours to accommodate slow-growing bacteria. Colonies were classified by morphology size, shape, and color, followed by subculture, until pure colonies formed.
Bacterial Gram-staining
The Gram staining process was carried out for identification of Gram-positive and Gram-negative bacterial colonies. Bacterial smears were prepared by mixing a small quantity of colony with a drop of sterile distilled water on a clean glass slide. Slides were allowed to air dry before being heated under a Bunsen burner flame. The Gram staining procedure involved washing in distilled water in between each application of crystal violet (1 minute), iodine solution (1 minute), 70% ethanol for decolorization (5 seconds), and safranin (1 minute). After air drying, stained slides were observed at 100 × magnification using an oil immersion light microscope. Gram-negative bacteria appeared pink or red, and Gram-positive bacteria were purple.
DNA extraction
Approximately 20 mg of bacterial pellet was added to a 1.5 ml centrifuge tube accordingly for centrifugation at 8000 x g for around 5 minutes to remove the supernatant. Then, the lysis process was carried out differently for both Gram-positive and Gram-negative bacteria. For Gram-positive bacteria and bacterial pellets were resuspended in a 180 μL bacteria pre-lysis buffer, followed by the addition of lysozyme with final concentration of 20 mg/mL. The samples were incubated for 30 minutes at 37 °C. Meanwhile, Gram-negative bacterial pellet, which was resuspended in 180 μL TLB1 buffer.
The following steps are similar for Gram-positive and Gram-negative bacterial DNA extraction. Then, 25 μL of Proteinase K solution was added to the tube, and it was vortexed vigorously [28]. Subsequently, all tubes were sealed using parafilm and incubated for 3 hours at 56 °C. The samples were vortexed every 15 minutes for the first two hours. After 3 hours, the samples were taken out from the incubator, followed by the addition of 200 μL of TLB2 buffer into it. The samples were then vortexed vigorously. Then, it was incubated at 70 °C for about 10 minutes. Afterwards, centrifugation was performed, containing insoluble particles at 11,000 x g. Then, the supernatant was transferred to a new 1.5 mL centrifuge tube.
The next step is the binding process. 210 μL of 100% ethanol was added and mixed by vortexing. Then, the lysate was transferred into a PrimeWay Genomic Column and was centrifuged at 11,000 x g for one minute. Subsequently, the PrimeWay Genomic Column was placed onto a new collection tube. The following step was washing the samples by adding 500 μL of Wash Buffer T1 into the column. Centrifugation was done with a condition of 11,000 x g for one minute. The flow-through was discarded and the genomic column was placed back into the collection tube. The same step was repeated by adding 600 μL Wash Buffer T2 into it. For the drying process, the samples were centrifuged again for one minute to remove ethanol residue. Then, the genomic column was placed in a new centrifuge tube. Finally, 50 μL of elution buffer was added to the center of the column membrane. The column was kept at room temperature for one minute and then followed by centrifugation to elute DNA.
PCR amplification and sequencing
All the PCR components were assembled in a 0.2 mL PCR tube with PCR components of sterile distilled water (13.5 μL), DreamTaq Mastermix (12.5 μL), reverse primer (1 μL), forward primer (1 μL) and DNA template (2 μL). To produce negative control, 2 μL of sterile distilled water was included to replace the DNA template. The 16S rRNA genes of their V3-V4 regions were amplified and sequenced on an Illumina HiSeq 2500 platform. The primer set used for this study consisted of a forward primer (27F), 5’ GAG TTT GAT CCT GGC TCA 3’, and a reverse primer (1492R), 5’ CGG CTA CCT TGT TAC GAC TT 3’. PCR amplification was performed using the T100 TM thermal cycler (Bio-Rad Laboratories, Inc., USA). The PCR cycling conditions were used started with initial denaturation at 95 °C for 1 minute, followed by denaturation at 95 °C for 30 seconds. Then, the annealing process took place at 50 °C for 30 seconds, and the extension was at 72 °C for one minute. Denaturation, annealing, and extension processes underwent 35 cycles in total. Finally, the final extension process occurred at 72 °C for five minutes (Lee et al., 2019). The PCR products were analyzed by agarose gel electrophoresis.
Analysis of sequencing data
After the base calling analysis, the original data files from the sequencing platform were transformed into the original sequenced reads stored in FASTQ format. All the sequences were submitted to the GenBank database. Sequences were analyzed using the Molecular Evolutionary Genetics Analysis (MEGA) software version 11. Biodiversity indices such as the Simpson and Shannon indices of total isolated bacteria from different species and various types of ATSBs from different individuals were analyzed.
Results
The results of bacterial species isolated from the midgut of female wild-strain Ae. aegypti and Ae. albopictus mosquitoes under control and ATSBs treated under the conditions shown in Table 1. It shows that Bacillus cereus (PQ661268), Bacillus albus (PQ661254), and Bacillus altitudinis (PQ661256) dominated the microbiota of control wild female Ae. aegypti. On the other hand, female Ae. albopictus showed a unique microbial community that was mainly made up of Microbacterium maritypicum (PQ669681), Acinetobacter nosocomialis (PQ669684), and Clostridium sporogenes (PQ669689). The result indicates a possibly pathogenic and more anaerobic bacterial composition. Both mosquito species were exposed to a variety of gram-negative bacteria through the use of mango-based ATSBs. Klebsiella pneumoniae (PQ661365), Klebsiella grimontii (PQ661258) and Klebsiella aerogenes (PQ661262) are all Enterobacteriaceae that are renowned for their opportunistic pathogenicity and environmental adaptability and were shown to be abundant in the gut microbiota of Ae. aegypti. Bacteria such as B. altitudinis (PQ669661), Stenotrophomonas maltophilia (PQ670061) and Micrococcus aloeverae (PQ669216) colonized wild female Ae. albopictus. Interestingly, B. altitudinis was prevalent in both species, indicating that mango ATSBs were used for selective enrichment.
Following exposure to Chrysanthemum ATSBs, Pseudomonas aeruginosa (PQ661368), Bacillus startosphericus (PQ661257) and Bacillus velezensis (PQ661260) dominated the gut profiles of wild female Ae. aegypti. Delftia lacustris (PQ669672), S. maltophilia (PQ670839) and Stenotrophomonas pavanii (PQ670082) were significantly enriched in the gut of Ae. albopictus. Meanwhile, P. aeruginosa (PQ661269), Pseudomonas protegens (PQ661250) and E. cloacae (PQ661278) were in the gut of Ae. aegypti treated with mixed ATSBs. The B. cereus (PQ671051, PQ671077) and Paenibacillus sp. (PQ669708) which are commonly found in soil and insect guts, were the two most prevalent bacterial taxa in wild female Ae. albopictus. Remarkably, P. aeruginosa appeared in both species, indicating that it continues to proliferate in mosquitoes treated with ATSBs.
Based on Table 2, Ae. aegypti harbored microbiome consisting of S. haemolyticus (PQ676526), B. amyloliquefaciens (PQ678966) and S. pasteuri (PQ678886) in the controlled laboratory females. On the other hand, M. maritypicum, K. aerogenes (PQ681134), and K. grimontii (PQ681135) were found in Ae. albopictus. Notably, M. maritypicum was shared by both species, indicating overlap in the gut microbiota maintained in the control lab strain mosquito. Significant changes in microbiota have been driven about by the sugar baits made from mangoes. Along with P. aeruginosa (PQ681124), B. cereus (PQ678965), B. tropicus (PQ678967) and B. subtilis (PQ678969) were the predominant bacteria in Ae. aegypti. The C. botulinum (PQ681125), S. maltophilia (PQ681126) and P. aeruginosa were all present in Ae. albopictus. The two species shared P. aeruginosa, suggesting that mango ATSBs facilitate an environment for gram-negative bacteria to colonize.
Ae. aegypti was a vector of L. iners (PQ678970), B. paramycoides (PQ678885) and C. indologenes (PQ675785) in the Chrysanthemum group. However, B. velezensis (PQ681129), C. aureum (PQ681128) and C. indologens (PQ681127) were linked to Ae. albopictus. After being exposed to Chrysanthemum ATSBs, both mosquito species showed the presence of Chryseobacterium spp., indicating that floral ATSBs play a major role in the acquisition of plant-associated bacteria. The Ae. aegypti mosquitoes treated with mixed ATSBs showed indications of B. cereus (PQ678956), Aeromonas veronii (PQ675812) and E. asburiae (PQ675780). The A. veronii (PQ681131), K. grimontii (PQ681130) and K. pneumoniae (PQ681132) were all present in Ae. albopictus. The ability of mixed floral baits to transfer water-associated opportunistic bacteria into the gut is suggested by the consistent finding of A. veronii in both species.
Table 3 shows that the bacterial population in the wild strain male Ae. aegypti control group was primarily made up of B. cereus (PQ661266, PQ661267) and B. tropicus (PQ661264). On the other hand, L. komagatae (PQ671410), B. tropicus (PQ671280) and B. cereus (PQ671709) were found in Ae. albopictus. Interestingly, both mosquito species shared B. cereus and B. tropicus, indicating that these bacterial taxa may be a natural component of the microbiota in wild male populations. Bacteria such as E. cloacae (PQ661157), B. amyloliquefaciens (PQ661377) and B. cereus (PQ661373) colonized Ae. aegypti treated with mango ATSBs. Similarly, E. coli (PQ668632) and E. cloacae (PQ668541, PQ670976) were repeatedly found in Ae. albopictus. Given that E. cloacae are frequently found in both species, it is likely due to the sugar-rich formulation of mango ATSBs favoring facultative anaerobes, which in turn promotes the growth or acquisition of enteric bacteria.
B. licheniformis (PQ661367), M. paraoxydans (PQ661363), and B. thuringiensis (PQ661366) were among the microbes that are present in Ae. aegypti exposed to Chrysanthemum ATSBs. In contrast, C. bernardetii (PQ668615), C. indologenes (PQ668617), and C. rhizoplanae (PQ668633) were linked to Ae. albopictus. This tendency suggests a divergence in bacterial uptake between species, such as wild male Ae. albopictus prefers Chryseobacterium spp., which are known to be prevalent in plant-associated settings. The microbiota of Ae. aegypti mosquitoes are treated with mixed ATSBs including A. viridans (PQ670297), B. thuringiensis (PQ661372, PQ661378) and B. paramycoides (PQ661364). Meanwhile, P. dispersa (PQ671643) and E. ludwigii (PQ669186) were found in Ae. albopictus. Notably, enteric bacteria like Pantoea and Enterobacter predominated in Ae. albopictus, but B. thuringiensis was frequently found in wild male Ae. aegypti.
According to Table 4, three different bacterial species were found in control male Ae. aegypti laboratory mosquitoes such as B. cereus (PQ678982), M. endophyticus (PQ678981) and B. thuringiensis (PQ678980). In contrast, A. variabilis (PQ681121), B. albus (PQ681122) and B. tropicus (PQ681123) dominated the bacterial profile of lab strain male Ae. albopictus. Under laboratory circumstances, there was no evidence of species overlapping between the two species in the untreated group, suggesting species-specific core microbiota. Microbial profiles of the two species showed both overlap and divergence as a result of exposure to mango ATSBs. Three bacterial species were identified in Ae. aegypti consists of B. sonorensis (PQ678973), C. botulinum (PQ678972), and B. subtilis (PQ678971). Nevertheless, B. thuringiensis (PQ681114) and B. cereus (PQ681112, PQ681113) dominated lab strain male Ae. albopictus. Interestingly, B. cereus was present in both mosquito species, indicating that the sugar bait media had an impact on bacterial colonization.
After being exposed to Chrysanthemum ATSBs, Ae. aegypti showed relatively diversified microbiota, including C. aureum (PQ681115), B. cereus (PQ678974, PQ678976), and M. luteus (PQ678975). The bacteria B. subtilis (PQ681116), C. indologenes (PQ681117), and C. aureum (PQ681115) were among the microbiota in Ae. albopictus midgut. The multiple occurrences of C. aureum indicate that some bacteria originating from flowers may be able to colonize different mosquito species. Significant taxonomic overlaps were seen in the microbiome of both mosquito species exposed to mixed ATSBs. B. cereus (PQ678977, PQ678978, and PQ678979) and Lysinibacillus fusiformis (PQ681118) were repeatedly isolated from Ae. aegypti. Along with B. cereus, Ae. albopictus also shows the presence of S. epidermidis (PQ681119) and S. warneri (PQ681120). The high prevalence of B. cereus in both species highlights its ecological competitiveness and resistance in sugar-rich habitats.
A total of 24 various bacteria species were found in Aedes mosquitoes that fed on mango ATSBs (Fig 1, S1 Table). All isolates were members of three phyla from Actinobacteria (4.16%), Firmicutes (54.17%), and Proteobacteria (41.67%). Micrococcus aloeverae from the phylum Actinobacteria, is only present in female ALW. Bacteria from wild strain female Ae. albopictus were from three different phyla such as Proteobacteria (Stenotrophomonas maltophilia), Firmicutes (Bacillus altitudinis) and Actinobacteria (Micrococcus aloeverae). The midgut of mango ATSBs fed laboratory strain male and female Ae. aegypti and male Ae. albopictus is fully colonized by phyla Firmicutes. Genus Bacillus dominated the midgut of these mosquitoes, followed by genus Clostridium. However, phyla Proteobacteria fully occupied the midgut of wild-strain female Ae. aegypti and male Ae. albopictus. The dominant bacterial species identified was Bacillus cereus (16.67%), followed by Enterobacter cloacae (12.5%).
According to Fig 1, bacterial species isolated from the midgut of Aedes mosquitoes that fed on Chrysanthemum ATSBs were from four different phyla. All isolates were members of Actinobacteria (8.33%), Firmicutes (41.67%), Bacteroidetes (33.33%), and Proteobacteria (16.67%). All the bacteria from phylum Firmicutes are under genus Bacillus (100%), while genus Chryseobacterium (100%) dominates phylum Bacteroidetes. The data shows that female ALW consists of bacteria from the phylum Proteobacteria, such as Delftia lacustris, Stenotrophomonas maltophilia, and Stenotrophomonas pavanii. However, male ALW only contains microbiomes from the phylum Bacteroidetes, such as C. bernadetii, C. indologenes, and C. rhizoplanae.
Furthermore, the microbiome isolated from the midgut of Aedes mosquitoes after exposure to mixed ATSBs indicate that there were only two phylum presents, which are Firmicutes (58.33%) and Proteobacteria (41.67%). Phylum Firmicutes consists of the genera Bacillus, Clostridium, and Staphylococcus, while phylum Proteobacteria consists of the genera Aeromonas, Pantoea, Enterobacter, Pseudomonas, and Klebsiella. The midgut of female AGW and ALL is completely colonized by the phylum Proteobacteria. In addition, male AGW, AGL, and ALL, including female ALW, are predominantly established by phylum Firmicutes. Among all the microbiomes, Bacillus cereus has the highest abundance, 20.83% in total. There are other Bacillus species found in the midgut of Aedes mosquitoes, such as Bacillus paramycoides and Bacillus thuringiensis.
The mosquitoes fed on control ATSBs confirm the presence of three different phyla, namely Proteobacteria (8.33%), Actinobacteria (25%), and Firmicutes (66.67%). Male AGW and female AGL only indicate the Firmicutes group bacteria (100%) in the midgut of the mosquitoes. Phylum Proteobacteria is only present in female ALL which is represented by Klebsiella grimontii and Pseudomonas aeruginosa. Examples of Phylum Actinobacteria that are found in the mosquito midgut are Microbacterium maritypicum, Micrococcus endophyticus, and Leucobacter komagatae. The genera Bacillus and Staphylococcus from the phylum Firmicutes are the dominant bacterial species group in the control ATSBs fed mosquito midgut.
A total of 48 distinct bacterial species were isolated and identified from the midgut-sampled mosquitoes. The most abundant species was Bacillus cereus (nᵢ = 17, pᵢ = 0.18), and some species were represented by only one or two isolates (pᵢ = 0.01–0.02). Using these relative abundances, the Shannon–Wiener diversity index was calculated as H′ = 6.68, and Simpson’s diversity index was D = 0.94 (Table 5). The higher H′ value indicates considerable species richness and evenness, and the higher D value (close to 1) shows the low dominance of any single species within the midgut bacterial community.
Discussion
Aedes mosquitoes are major vectors of arboviral diseases and pose a substantial public health threat [29–31]. Consequently, their midgut microbiome particularly in the context of different sugar sources is highly relevant for understanding microbiota, vector and pathogen interactions, as shifts in bacterial communities can influence vector competence, survival, physiology, and overall ecological fitness of mosquito populations [28,32] (Lin et al., 2021; Yadav et al., 2015). In this study, clear differences were observed in the composition of bacterial taxa before and after exposure to the various sugar treatments, indicating that diet can drive measurable restructuring of the midgut microbiota. Most bacteria recovered from mango and mixed treatments belonged to the family Enterobacteriaceae, with Klebsiella and Enterobacter representing the most abundant genera, including species such as Klebsiella pneumoniae, Klebsiella grimontii, Enterobacter ludwigii, Enterobacter cloacae, and Enterobacter asburiae. These findings agree with previous work showing that Enterobacter spp. are among the most frequently isolated bacteria from Aedes mosquitoes [13,28,32].
The detection of E. cloacae is particularly noteworthy because multiple studies have demonstrated its capacity to inhibit Plasmodium development in the midgut of Anopheles mosquitoes, via immune activation and reactive oxygen species–mediated antiparasitic effects [33,34]. Enterobacter cloacae is widely regarded as a common gut symbiont that can modulate mosquito physiology and vector competence and has therefore been explored as a candidate for paratransgenic approaches, in which it is engineered to express anti-pathogen effector molecules to reduce transmission [33]. Beyond mosquitoes, E. cloacae has successfully been used in paratransgenesis to control Leishmania within the sand fly Phlebotomus papatasi [35] and has been shown to transfer and express foreign genes in termite colonies, demonstrating its potential as a versatile symbiotic chassis [36]. Collectively, these data suggest that E. cloacae either in its native form or as a paratransgenic platform could be exploited to control vector-borne pathogens and reduce disease transmission.
Since Klebsiella is a frequently reported bacterial genus in the midgut of Aedes mosquitoes, its detection in this study is also notable. Klebsiella spp., particularly K. pneumoniae and K. oxytoca, are recognized gut symbionts in mosquitoes and have been implicated in host development, immune modulation, and interference with pathogen infection. A previous study investigated the symbiotic interaction between Ae. aegypti and Klebsiella spp. and showed that volatile organic compounds (VOCs) released by Klebsiella strains significantly affected the oviposition choices of gravid females [37]. These VOCs influenced the selection of breeding sites and may therefore alter population dynamics and the transmission of mosquito-borne pathogens. Such findings suggest that incorporating oviposition-stimulating VOCs into “attract-and-kill” systems could be a promising strategy for mosquito management, by luring gravid females to lethal oviposition sites that combine attractive cues with targeted killing agents [5].
Members of the gut microbiota can contribute to nutrient metabolism, including the processing of nitrogenous compounds, and thereby support mosquito larval development and adult fecundity. Several gut-associated bacteria, including Klebsiella spp., have been implicated in vitamin and amino-acid provisioning and in enhancing growth and survival in mosquitoes and other insects. In addition, commensal bacteria are known to stimulate the mosquito immune system, leading to increased production of antimicrobial peptides and reactive oxygen species that can limit the establishment or development of pathogens such as Plasmodium and arboviruses [24].
Owing to their capacity to colonize the mosquito gut and to modulate both host physiology and infection outcomes, Klebsiella spp. have been explored as paratransgenic candidates for vector control, engineered to express anti-pathogen effector molecules within the midgut environment [15,38]. Beyond mosquitoes, Klebsiella pneumoniae has been evaluated as a biotechnological tool for insect control and sterilization, for example in strategies targeting the Mediterranean fruit fly, and has also been reported as an endosymbiont in grasshoppers [39,40].
Numerous bacterial genera including Enterobacter, Klebsiella, Pantoea, Acinetobacter,Pseudomonas,Bacillus,Staphylococcus,Micrococcus and Aeromons have been repeatedly isolated from the midgut of different mosquito species and are also represented among the taxa detected in this study [41–43]. In particular, previous work on the midgut microbiota of Ae. aegypti and Ae. albopictus collected from geographically distinct locations has recovered species such as Aeromonas veronii, Bacillus aerophilus, Bacillus cereus, Enterobacter asburiae, Enterobacter cloacae, Klebsiella pneumoniae, Pantoea dispersa, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia [32], supporting the view that many of these bacteria establish themselves as commensal microbiota and play important roles in mosquito development, physiology, and vector competence [44].
The composition of the mosquito midgut microbiota is shaped by multiple intrinsic and extrinsic factors, including sex, strain origin, and diet. Previous work has shown that mosquito gender is associated with differences in midgut bacterial diversity [41], and our observations are consistent with this pattern. Several bacterial taxa in our study were detected exclusively in females (e.g., Micrococcus aloeverae, Staphylococcus haemolyticus, Staphylococcus pasteuri, Aeromonas veronii, Microbacterium maritypicum), whereas others occurred only in males (e.g., Clostridium botulinum, Micrococcus endophyticus, Staphylococcus warneri, Lysinibacillus fusiformis, Acinetobacter variabilis). Microbial diversity was generally higher in females across species and strains, corroborating earlier findings that link increased bacterial richness to hematophagous behaviour and associated ecological exposures [45]. Female mosquitoes encounter a wider range of microbial sources through blood feeding and oviposition, which may facilitate acquisition of gut-associated opportunists such as Clostridium, Chryseobacterium and Acinetobacter spp., whereas males, which rely primarily on sugar meals, tend to harbour communities dominated by Bacillus and other Firmicutes, reflecting more restricted dietary and environmental interactions [46,47].
Strain origin further structured the microbiota. Wild-strain mosquitoes of both sexes carried a broader range of taxa, including Paenibacillus, Aeromonas, Enterobacter and Acinetobacter spp., consistent with a strong influence of environmental exposure to natural habitats and organic substrates [7]. Paenibacillus is notable for its ability to produce antimicrobial peptides and fix nitrogen, traits that may contribute to nutritional homeostasis and pathogen modulation in the mosquito gut [48]. In contrast, laboratory strains showed reduced bacterial diversity, with profiles dominated largely by Firmicutes, in line with previous reports that standardized diets, controlled conditions, and limited environmental microbial reservoirs constrain microbiome complexity [49].
Across treatments, members of the genus Bacillus particularly B. cereus, B. thuringiensis and B. subtilis were frequently detected in both Aedes species and sexes. The prominence of Bacillus spp. agrees with earlier studies identifying spore-forming Firmicutes as common mosquito gut symbionts, likely due to their environmental resilience and metabolic versatility [50]. These bacteria have been associated with enhanced tolerance to environmental stress and improved digestive enzyme activity in the gut [15], while B. thuringiensis is well known for producing insecticidal toxins used in larvicidal control [51]. Klebsiella spp. (e.g., K. pneumoniae, K. grimontii, K. aerogenes) were particularly prevalent in females exposed to mango-based ATSBs, suggesting acquisition from floral or environmental sources. In wild females, Pseudomonas aeruginosa and Stenotrophomonas maltophilia were also frequent, including after ATSB exposure, possibly reflecting adaptive colonization of sugar-rich midgut environments or residual microbes from plant baits [52].
Floral-based sugar baits markedly altered midgut community composition. Mango ATSBs favoured colonization by facultative anaerobes such as Klebsiella, Clostridium and Enterobacter, taxa known for their metabolic adaptability and ability to utilize diverse carbohydrate substrates [53]. Chrysanthemum ATSBs increased the relative abundance of Chryseobacterium, Stenotrophomonas and Pseudomonas spp., genera often associated with environmental and plant microbiota and known to produce antimicrobial metabolites [50]. Mixed ATSBs supported the broadest bacterial spectrum, including Paenibacillus, Aeromonas and Pantoea spp., suggesting that composite floral blends may enhance microbiome richness via nutritional diversity or facilitation among bacterial taxa. In addition, several less-commonly reported species such as Chryseobacterium rhizoplanae, Microbacterium paraoxydans and Clostridium sporogenes were detected. C. rhizoplanae was originally described from maize rhizoplanes [54], M. paraoxydans has been reported from clinical and aquatic sources [55,56], and C. sporogenes is known from mammalian gastrointestinal tracts [57], indicating that mosquitoes may act as incidental carriers of environmental and vertebrate-associated bacteria.
Diversity metrics in our data (e.g., high Shannon index and Simpson’s evenness) point to a complex and relatively balanced midgut community. Similar levels of diversity have been reported in other mosquito microbiome studies and have been linked to host physiological and immunological functions [6,28,58]. The dominance of B. cereus may indicate a competitive advantage under midgut conditions, yet the presence of many low-abundance taxa suggests a substantial “rare biosphere” contributing to functional redundancy and resilience. High microbial diversity is often associated with greater stability of insect gut communities and may help maintain host homeostasis under fluctuating environmental conditions [58]. In mosquitoes, midgut bacteria participate in digestion, nutrient assimilation, immune priming, and modulation of vector competence [6,59], implying that the rich communities observed here could have important, multifaceted effects on host biology.
Functionally, ATSB-induced shifts in microbiome composition may have major implications for mosquito physiology and vectorial capacity. By altering the nutrient and chemical landscape of the midgut, floral sugar baits impose selective pressures that favour bacteria with specific metabolic traits [52,53]. The predominance of Enterobacteriaceae in sugar-rich treatments suggests that these diets promote fermentative and nitrogen-cycling bacteria, which can improve host nutritional efficiency and potentially influence fecundity and longevity [7,15,48] Furthermore, distinct floral baits likely reshape gut ecology through differential availability of sugars, secondary metabolites and antimicrobial phytochemicals, leading to changes in competition and colonization patterns [53]. Modifications in gut pH, osmolarity and immune activation triggered by these diets may also modulate the abundance of key taxa such as Enterobacter and Klebsiella, which can stimulate immune pathways that produce antimicrobial peptides and reactive oxygen species able to inhibit pathogen development [15,28,33]. In this way, enrichment of immunomodulatory symbionts through ATSB exposure could reduce vector competence while simultaneously enhancing mosquito resilience to environmental stress.
From a vector-control perspective, the observed microbiome responses highlight opportunities to optimize ATSB formulations. Floral baits that promote beneficial symbionts particularly E. cloacae, which has documented pathogen-blocking capacities could be combined with paratransgenic approaches to deliver anti-pathogen effectors and enhance control efficacy [33,35]. Mixed-floral ATSBs, which appear to increase microbial diversity, may help sustain protective microbial communities that bolster immune defences and lower arboviral transmission potential [7,52]. Altogether, our results indicate that ATSB exposure not only modifies mosquito feeding behaviour but also drives substantial restructuring of the midgut microbiota, with cascading consequences for physiology, immunity and vector competence. This intimate link between diet, microbiome composition and pathogen interference underscores the potential for integrating microbiome-based interventions with ecologically informed sugar-bait strategies to develop sustainable, next-generation mosquito control programmes [7,28].
Methodologically, this study used pooled midgut samples (five individuals per replicate) to reduce inter-individual variation and secure sufficient DNA yields for sequencing, an approach supported by previous mosquito microbiome work [60,61]. The 72-h post-ATSB exposure time point was selected based on earlier studies showing pronounced microbiome shifts within this window [60]. Future investigations incorporating larger sample sizes, multiple time points and single-mosquito analyses will be important to refine our understanding of the temporal dynamics and individual variability of gut microbial responses to ATSB exposure.
Conclusion
This study characterized the abdominal microbiota composition and midgut and ovary histology in Aedes aegypti exposed to hydrogel baits infused with chrysanthemum, mango, mixed sugar, or sucrose control. Sanger sequencing revealed Proteobacteria dominance across all treatments, with Enterobacteriaceae comprising 90% of chrysanthemum-associated taxa and prominent in mixed sugar treatments, Lactobacillaceae (40%) unique to mango treatments, and Alphaproteobacteria (75%) predominant in sucrose controls. Histological analysis identified sugar variant specific midgut epithelial changes: prominent cell breakage in mixed sugar treatments versus minor disruption and clearing in mango/chrysanthemum treatments. Ovaries exhibited slight follicular cell displacement across all groups.
Several limitations constrain interpretation: (1) absence of extraction blanks precludes definitive exclusion of low-level contamination despite negative no-template PCR controls; (2) direct Sanger sequencing provides limited community resolution compared to high-throughput 16S rRNA or metagenomic approaches; (3) dataset structure did not permit robust beta-diversity analyses, restricting comparisons to descriptive taxonomy and basic histopathological observations.
These preliminary findings from dengue endemic Malaysia demonstrate that hydrogel sugar baits induce detectable microbiota shifts and midgut histopathological changes in Ae. aegypti. Further studies with comprehensive controls and advanced sequencing are required to confirm these observations and elucidate biological implications.
Supporting information
S1 Table. Bacterial species detected in male and female Aedes aegypti and Aedes albopictus (wild and laboratory strains) treated with different sugar variants.
https://doi.org/10.1371/journal.pone.0329341.s001
(DOCX)
S1 File. Inclusivity-in-global-research-questionnaire.
https://doi.org/10.1371/journal.pone.0329341.s002
(DOCX)
Acknowledgments
We would like to thank the Vector Control Research Units (VCRU) for egg supplies and BioG Expert SDN. BHD. for hydrogel beads preparation facilities. This work was supported by Ministry of Higher Education Malaysia, Fundamental Research Grant Scheme (FRGS) with Reference No: FGRS/1/2022/STG03/USM/02/8.
References
- 1. Abuelmaali SA, Jamaluddin JAF, Allam M, Abushama HM, Elnaiem DE, Noaman K, et al. Genetic polymorphism and phylogenetics of aedes aegypti from sudan based on ND4 Mitochondrial gene variations. Insects. 2022;13(12):1144. pmid:36555054
- 2. Noaman K, Abuelmaali SA, Elnour M-AB, Korti M, Ageep T, Baleela RMH. First detection of F1534C kdr insecticide resistance mutation in Aedes aegypti in Sudan. Parasitol Res. 2024;123(4):178. pmid:38578300
- 3. Mashlawi AM, Alqahtani H, Abuelmaali SA, Gloria-Soria A, Saingamsook J, Kaddumukasa M, et al. Microsatellite-based analysis reveals Aedes aegypti populations in the Kingdom of Saudi Arabia result from colonization by both the ancestral African and the global domestic forms. Evol Appl. 2024;17(2):e13661. pmid:38405337
- 4. Sambanthan R, Abu Kassim NF, Abuelmaali SA, Wan Ahmad Kamil WM, Sabar S, Zarkasi KZ, et al. Formulation and testing of different infused attractive sugar baits (ASBs) in sodium alginate against laboratory strain Aedes aegypti. Exp Parasitol. 2025;277:109026. pmid:40957531
- 5. Ayub NM, Kassim NFA, Sabar S, Hashim NA, Lalung J, Sulaiman SF, et al. Evaluation of fruit combinations as potential liquid attractants for hydrogel bait applications targeting Aedes mosquitoes. Parasit Vectors. 2026;19(1):69. pmid:41491233
- 6. Dong Y, Manfredini F, Dimopoulos G. Implication of the mosquito midgut microbiota in the defense against malaria parasites. PLoS Pathog. 2009;5(5):e1000423. pmid:19424427
- 7. Minard G, Mavingui P, Moro CV. Diversity and function of bacterial microbiota in the mosquito holobiont. Parasit Vectors. 2013;6:146. pmid:23688194
- 8. Zhang R, Zhao T, Xing D, Zhou X, Yu H, Geng D, et al. Development of Attractive Toxic Sugar Baits (ATSBs) System and Its Effectiveness in Mosquito Control. Insects. 2025;16(3):258. pmid:40266766
- 9.
Sambanthan R, Kassim NFA, Abuelmaali SA, Kamil WMWA, Sabar S, Zarkasi KZ, et al. Impact of olfactory sensitivity preferences on midgut pathophysiology of Aedes mosquitoes towards different sugar variants. Springer Science and Business Media LLC; 2025. https://doi.org/10.21203/rs.3.rs-7141836/v1
- 10.
National Institute of Environmental Health Sciences. Microbiome. 2025. [Cited 2025 July 10]. https://www.niehs.nih.gov/health/topics/science/microbiome
- 11. Villegas LEM, Radl J, Dimopoulos G, Short SM. Bacterial communities of Aedes aegypti mosquitoes differ between crop and midgut tissues. PLoS Negl Trop Dis. 2023;17(3):e0011218. pmid:36989328
- 12. Scolari F, Casiraghi M, Bonizzoni M. Aedes spp. and their microbiota: A review. Front Microbiol. 2019;10:2036. pmid:31551973
- 13. David MR, Santos LMBD, Vicente ACP, Maciel-de-Freitas R. Effects of environment, dietary regime and ageing on the dengue vector microbiota: evidence of a core microbiota throughout Aedes aegypti lifespan. Mem Inst Oswaldo Cruz. 2016;111(9):577–87. pmid:27580348
- 14. Weiss B, Aksoy S. Microbiome influences on insect host vector competence. Trends Parasitol. 2011;27(11):514–22. pmid:21697014
- 15. Gusmão DS, Santos AV, Marini DC, Bacci M Jr, Berbert-Molina MA, Lemos FJA. Culture-dependent and culture-independent characterization of microorganisms associated with Aedes aegypti (Diptera: Culicidae) (L.) and dynamics of bacterial colonization in the midgut. Acta Trop. 2010;115(3):275–81. pmid:20434424
- 16. Ramirez JL, Souza-Neto J, Torres Cosme R, Rovira J, Ortiz A, Pascale JM, et al. Reciprocal tripartite interactions between the Aedes aegypti midgut microbiota, innate immune system and dengue virus influences vector competence. PLoS Negl Trop Dis. 2012;6(3):e1561. pmid:22413032
- 17. Chun J, Rainey FA. Integrating genomics into the taxonomy and systematics of the Bacteria and Archaea. Int J Syst Evol Microbiol. 2014;64(Pt 2):316–24. pmid:24505069
- 18. Fukuda K, Ogawa M, Taniguchi H, Saito M. Molecular approaches to studying microbial communities: targeting the 16S Ribosomal RNA gene. J UOEH. 2016;38(3):223–32. pmid:27627970
- 19. Yang M-Q, Wang Z-J, Zhai C-B, Chen L-Q. Research progress on the application of 16S rRNA gene sequencing and machine learning in forensic microbiome individual identification. Front Microbiol. 2024;15:1360457. pmid:38371926
- 20. Salgado JFM, Premkrishnan BNV, Oliveira EL, Vettath VK, Goh FG, Hou X, et al. The dynamics of the midgut microbiome in Aedes aegypti during digestion reveal putative symbionts. PNAS Nexus. 2024;3(8):pgae317. pmid:39157462
- 21. Goodman AM, Castro A, Pyke RM, Okamura R, Kato S, Riviere P, et al. MHC-I genotype and tumor mutational burden predict response to immunotherapy. Genome Med. 2020;12(1):45. pmid:32430031
- 22. Almire F, Terhzaz S, Terry S, McFarlane M, Gestuveo RJ, Szemiel AM, et al. Sugar feeding protects against arboviral infection by enhancing gut immunity in the mosquito vector Aedes aegypti. PLoS Pathog. 2021;17(9):e1009870. pmid:34473801
- 23. Ni CY, Kassim NFA, Ayub NM, Abuelmaali SA, Mashlawi AM, Dieng H. Impact of diverse musical genres on blood-feeding and mating behavior in Aedes aegypti mosquitoes. J Vector Borne Dis. 2025;62(2):211–7. pmid:39636255
- 24. Suruchi S, Harsh R, Varsha S, Raghav D, Taniya G, Meenakshi T. Alginate based Nanoparticles and Its application in drug delivery systems. Journal of Pharmaceutical Negative Results. 2022:1463–9.
- 25. Lee KY, Mooney DJ. Alginate: properties and biomedical applications. Prog Polym Sci. 2012;37(1):106–26. pmid:22125349
- 26. McCalla KA, Tay J-W, Mulchandani A, Choe D-H, Hoddle MS. Biodegradable alginate hydrogel bait delivery system effectively controls high-density populations of Argentine ant in commercial citrus. J Pest Sci. 2020;93(3):1031–42.
- 27. Tay J-W, Choe D-H, Mulchandani A, Rust MK. Hydrogels: From controlled release to a new bait delivery for insect pest management. J Econ Entomol. 2020;113(5):2061–8. pmid:32852040
- 28. Lin D, Zheng X, Sanogo B, Ding T, Sun X, Wu Z. Bacterial composition of midgut and entire body of laboratory colonies of Aedes aegypti and Aedes albopictus from Southern China. Parasit Vectors. 2021;14(1):586. pmid:34838108
- 29. Abuelmaali SA, Jamaluddin JAF, Noaman K, Allam M, Abushama HM, Elnaiem DE, et al. Distribution and Genetic Diversity of Aedes aegypti Subspecies across the Sahelian Belt in Sudan. Pathogens. 2021;10(1):78. pmid:33477339
- 30. Sambanthan R, Kamarudin ANA, Ayub NM, Abuelmaali SA, Abu Kassim NF. Predicting dengue outbreak risk using epidemiological parameters related to the knowledge and awareness of community in selangor on dengue during Covid-19 pandemic. Malays J Med Health Sci. 2024;20(5):174–83.
- 31. Abuelmaali SA, Mashlawi AM, Ishak IH, Wajidi MFF, Jaal Z, Avicor SW, et al. Population genetic structure of Aedes aegypti subspecies in selected geographical locations in Sudan. Sci Rep. 2024;14(1):2978. pmid:38316804
- 32. Yadav KK, Bora A, Datta S, Chandel K, Gogoi HK, Prasad GBKS, et al. Molecular characterization of midgut microbiota of Aedes albopictus and Aedes aegypti from Arunachal Pradesh, India. Parasit Vectors. 2015;8:641. pmid:26684012
- 33. Cirimotich CM, Dong Y, Clayton AM, Sandiford SL, Souza-Neto JA, Mulenga M, et al. Natural microbe-mediated refractoriness to Plasmodium infection in Anopheles gambiae. Science. 2011;332(6031):855–8. pmid:21566196
- 34. Ezemuoka LC, Akorli EA, Aboagye-Antwi F, Akorli J. Mosquito midgut enterobacter cloacae and serratia marcescens affect the fitness of adult female anopheles gambiae s.l. PLOS ONE. 2020;15(9):e0238931.
- 35. Machado ART, Ferreira SR, da Silva Medeiros F, Fujiwara RT, de Souza Filho JD, Pimenta LPS. Nematicidal activity of Annona crassiflora leaf extract on Caenorhabditis elegans. Parasit Vectors. 2015;8:113. pmid:25885032
- 36. Husseneder C, Grace JK. Genetically engineered termite gut bacteria (Enterobacter cloacae) deliver and spread foreign genes in termite colonies. Appl Microbiol Biotechnol. 2005;68(3):360–7. pmid:15742168
- 37. Mosquera KD, Martinez Villegas LE, Pidot SJ, Sharif C, Klimpel S, Stinear TP, et al. Multi-Omic analysis of symbiotic bacteria associated with aedes aegypti breeding sites. Front Microbiol. 2021;12:703711. pmid:34475861
- 38. Tchioffo MT, Boissière A, Abate L, Nsango SE, Bayibéki AN, Awono-Ambéné PH, et al. Dynamics of bacterial community composition in the malaria mosquito’s Epithelia. Front Microbiol. 2016;6:1500. pmid:26779155
- 39. Kyritsis GA, Augustinos AA, Cáceres C, Bourtzis K. Medfly Gut Microbiota and Enhancement of the Sterile Insect Technique: Similarities and Differences of Klebsiella oxytoca and Enterobacter sp. AA26 Probiotics during the Larval and Adult Stages of the VIENNA 8D53+ genetic sexing strain. Front Microbiol. 2017;8:2064. pmid:29163379
- 40. Shi W, Uzuner U, Huang L, Jesudhasan PR, Pillai SD, Yuan JS. Comparative analysis of insect gut symbionts for composition–function relationships and biofuel application potential. Biofuels. 2011;2(5):529–44.
- 41. Valiente Moro C, Tran FH, Raharimalala FN, Ravelonandro P, Mavingui P. Diversity of culturable bacteria including Pantoea in wild mosquito Aedes albopictus. BMC Microbiol. 2013;13:70. pmid:23537168
- 42. Boissière A, Tchioffo MT, Bachar D, Abate L, Marie A, Nsango SE, et al. Midgut microbiota of the malaria mosquito vector Anopheles gambiae and interactions with Plasmodium falciparum infection. PLoS Pathog. 2012;8(5):e1002742. pmid:22693451
- 43. Zouache K, Raharimalala FN, Raquin V, Tran-Van V, Raveloson LHR, Ravelonandro P, et al. Bacterial diversity of field-caught mosquitoes, Aedes albopictus and Aedes aegypti, from different geographic regions of Madagascar. FEMS Microbiol Ecol. 2011;75(3):377–89. pmid:21175696
- 44. Chandel K, Mendki MJ, Parikh RY, Kulkarni G, Tikar SN, Sukumaran D, et al. Midgut microbial community of Culex quinquefasciatus mosquito populations from India. PLoS One. 2013;8(11):e80453. pmid:24312223
- 45. Dennison NJ, Saraiva RG, Cirimotich CM, Mlambo G, Mongodin EF, Dimopoulos G. Functional genomic analyses of Enterobacter, Anopheles and Plasmodium reciprocal interactions that impact vector competence. Malar J. 2016;15(1):425. pmid:27549662
- 46. Frankel-Bricker J. Shifts in the microbiota associated with male mosquitoes (Aedes aegypti) exposed to an obligate gut fungal symbiont (Zancudomyces culisetae). Sci Rep. 2020;10(1):12886. pmid:32733002
- 47. Strand MR. Composition and functional roles of the gut microbiota in mosquitoes. Curr Opin Insect Sci. 2018;28:59–65. pmid:30551768
- 48. Yang L, Quan X, Xue B, Goodwin PH, Lu S, Wang J, et al. Isolation and identification of Bacillus subtilis strain YB-05 and its antifungal substances showing antagonism against Gaeumannomyces graminis var. tritici. Biological Control. 2015;85:52–8.
- 49. Kamiya T, O’Dwyer K, Westerdahl H, Senior A, Nakagawa S. A quantitative review of MHC-based mating preference: the role of diversity and dissimilarity. Mol Ecol. 2014;23(21):5151–63. pmid:25251264
- 50. Ruiu L. Insect Pathogenic Bacteria in Integrated Pest Management. Insects. 2015;6(2):352–67. pmid:26463190
- 51. Sarrafzadeh MH, Guiraud JP, Lagneau C, Gaven B, Carron A, Navarro J-M. Growth, sporulation, delta-endotoxins synthesis, and toxicity during culture of bacillus thuringiensis H14. Curr Microbiol. 2005;51(2):75–81. pmid:16059772
- 52. Patil CD, Patil SV, Salunke BK. Influence of floral-based sugar sources on mosquito gut microbiota. Microbiological Research. 2022;256:126936.
- 53. Graf K, Ulrich A, Idler C, Klocke M. Bacterial community dynamics during ensiling of perennial ryegrass at two compaction levels monitored by terminal restriction fragment length polymorphism. J Appl Microbiol. 2016;120(6):1479–91. pmid:26923533
- 54. Kämpfer P, McInroy JA, Glaeser SP. Chryseobacterium rhizoplanae sp. nov., isolated from the rhizoplane environment. Antonie Van Leeuwenhoek. 2015;107(2):533–8. pmid:25515412
- 55. Laffineur K, Avesani V, Cornu G, Charlier J, Janssens M, Wauters G, et al. Bacteremia due to a novel Microbacterium species in a patient with leukemia and description of Microbacterium paraoxydans sp. nov. J Clin Microbiol. 2003;41(5):2242–6. pmid:12734292
- 56. Soto-Rodriguez SA, Cabanillas-Ramos J, Alcaraz U, Gomez-Gil B, Romalde JL. Identification and virulence of Aeromonas dhakensis, Pseudomonas mosselii and Microbacterium paraoxydans isolated from Nile tilapia, Oreochromis niloticus, cultivated in Mexico. J Appl Microbiol. 2013;115(3):654–62. pmid:23758410
- 57. Oakeson KF, Miller A, Dale C, Dearing D. Draft genome sequence of an oxalate-degrading strain of clostridium sporogenes from the gastrointestinal tract of the white-throated woodrat (Neotoma albigula). Genome Announc. 2016;4(3):e00392-16. pmid:27198026
- 58. Liu H, Yin J, Huang X, Zang C, Zhang Y, Cao J, et al. Mosquito gut microbiota: a review. Pathogens. 2024;13(8):691. pmid:39204291
- 59. Rajendran D, Vinayagam S, Sekar K, Bhowmick IP, Sattu K. Symbiotic bacteria: wolbachia, midgut microbiota in mosquitoes and their importance for vector prevention strategies. Microb Ecol. 2024;87(1):154. pmid:39681734
- 60. Dada N, Jupatanakul N, Minard G, Short SM, Akorli J, Villegas LM. Considerations for mosquito microbiome research from the Mosquito Microbiome Consortium. Microbiome. 2021;9(1):36. pmid:33522965
- 61. Cantú-Bernal S, Domínguez-Gámez M, Medina-Peraza I, Aros-Uzarraga E, Ontiveros N, Flores-Mendoza L, et al. Enhanced viability and anti-rotavirus effect of bifidobacterium longum and lactobacillus plantarum in combination with chlorella sorokiniana in a dairy product. Front Microbiol. 2020;11:875. pmid:32477300