The increasing global burden of mosquito-borne diseases and the widespread development of insecticide resistance in mosquitoes have fueled renewed interest in entomopathogenic fungi as effective tools that are compatible with existing mosquito control strategies. These fungi produce different types of infective propagules, including hydrophobic conidia and yeast-like blastospores, which differ in structure, mode of infection, and virulence. In this study, we evaluated the larvicidal activity of conidial and blastospore propagules from Beauveria bassiana MBC076 and Beauveria brongniartii MBC397 against Aedes aegypti. Conidia exhibited more rapid and more potent larvicidal effects compared to blastospores, but the overall survival at seven days post-infection was similar between the two types of propagules. Interestingly, B. brongniartii blastospore infections resulted in a significantly higher proportion of pupal mortality, suggesting a delayed mode of action. Immune profiling of infected larvae indicated significant induction of antimicrobial effectors such as cecropin, defensin, and attacin, primarily in response to conidial infection. In contrast, blastospore infections were associated with reduced expression of several prophenoloxidase genes, particularly during infection with B. brongniartii blastospores. These findings indicate that different fungal species and their propagule types exert varying levels of virulence and immune modulation in mosquito larvae. This study provides insights into the infection dynamics of fungal propagules and identifies immune markers that can be leveraged to enhance the efficacy of fungal-based larvicides.

Ticks are a medically important group of arthropods, and their control has become a major challenge due to their widespread resistance to synthetic acaricides. Defatted seed meals of plants in the mustard family, commonly generated as byproducts of vegetable oil and biofuel production are known to possess pesticidal properties but their potential to control ticks remain poorly understood. We evaluated the bio-fumigation activity of defatted seed meals of three mustard family species, Brassica juncea (L) Czern. (PG), Lepidium sativum L. (Ls) and Thlaspi arvense L. (DFP), against three medically important hard tick species: Ambylomma americanum (L.) [Aa], Dermacentor variabilis (Say) [Dv] and Ixodes scapularis (Say) [Is]. Volatiles produced by defatted seed meals of the three plant species had strong bio-fumigation activity against ticks, but their effects varied among the three tick species. Toxicity of DFP seed meals was significantly different among the three tick species (no overlap of the 95

Paenibacillus is a large genus of Gram-positive bacteria isolated from a wide range of sources. Members of this genus have attracted significant research as plant growth promoters as well as pathogens of both plants and animals. Extensive studies have been conducted on Paenibacillus larvae, the causative agent of American Foul Brood in honeybees, but little is known about other closely related species associated with honeybees, such as Paenibacillus apiarius or Paenibacillus thiaminolyticus. These bacterial species have been isolated from dead honeybee larvae and are believed to be secondary invaders as opposed to primary pathogens. We utilized genome mining efforts to explore the number and diversity of toxins present in multiple species of Paenibacillus, with major emphasis on a potentially entomopathogenic clade that includes Paenibacillus alvei, P. apiarius, and P. thiaminolyticus. Our results show that strains of these species contain a variety of potentially insecticidal toxins, including highly conserved Spp-like and Mpp-like toxins. These toxins are absent in P. larvae. We also found several ADP-ribosylases which were shared between P. larvae and several strains from this entomopathogenic clade. Tc toxins, a class of insecticidal toxin not often found in Gram-positive bacteria were found in in P. apiarius and P. alvei. These findings suggest the potential for Paenibacillus species in this clade to be harnessed as sources of host specific and pollinator-friendly biopesticides.

An isolate of a Gram-positive, strictly aerobic, motile, rod-shaped, endospore forming bacterium was originally isolated from soil when screening and bioprospecting for plant beneficial microorganisms. Phylogenetic analysis of the 16S rRNA gene sequences indicated that this strain was closely related to Lysinibacillus fusiformis NRRL NRS-350T (99.7

Container aquatic habitats host a community of aquatic insects, primarily mosquito larvae that browse on container surface microbial biofilm and filter-feed on microorganisms in the water column. We examined how the bacterial communities in these habitats respond to feeding by larvae of two container-dwelling mosquito species, Culex pipiens and Cx. restuans. We also investigated how the microbiota of these larvae is impacted by intra- and interspecific interactions. Microbial diversity and richness were significantly higher in water samples when mosquito larvae were present, and in Cx. restuans compared to Cx. pipiens larvae. Microbial communities of water samples clustered based on the presence or absence of mosquito larvae and were distinct from those of mosquito larvae. Culex pipiens and Cx. restuans larvae harbored distinct microbial communities when reared under intraspecific conditions and similar microbial communities when reared under interspecific conditions. These findings demonstrate that mosquito larvae play a major role in structuring the microbial communities in container habitats and that intra- and interspecific interactions in mosquito larvae may shape their microbiota. This has important ecological and public health implications since larvae of the two mosquito species are major occupants of container habitats while the adults are vectors of West Nile virus.

Accurate knowledge of blood meal hosts of different mosquito species is critical for identifying potential vectors and establishing the risk of pathogen transmission. We compared the performance of Miseq next generation sequencing approach relative to conventional Sanger sequencing approach in identification of mosquito blood meals using genetic markers targeting the 12S rRNA and cytochrome oxidase I (COI) genes. We analysed the blood meals of three mosquito vector species (Aedes aegypti, Aedes simpsoni s.l. and Culex pipiens s.l.) collected outdoors, and compared the frequency of single- versus multiple-blood feeding. Single host blood meals were mostly recovered for Sanger-based sequencing of the mitochondrial 12S rRNA gene, whereas Miseq sequencing employing this marker and the COI marker detected both single and multiple blood meal hosts in individual mosquitoes. Multiple blood meals (two or more hosts) which mostly included humans were detected in 19%-22.7% of Ae. aegypti samples. Most single host blood meals for this mosquito species were from humans (47.7%-57.1%) and dogs (9.1%-19.0%), with livestock, reptile and rodent hosts collectively accounting for 4.7%-28.9% of single host blood meals. The frequency of two or more host blood meals in Ae. simpsoni s.l. was 26.3%-45.5% mostly including humans, while single host blood meals were predominantly from humans (31.8%-47.4%) with representation of rodent, reptile and livestock blood meals (18.2%-68.2%). Single host blood meals from Cx. pipiens s.l. were mostly from humans (27.0%-39.4%) and cows (11.5%-27.36%). Multiple blood meal hosts that mostly included humans occurred in 21.2%-24.4% of Cx. pipiens s.l. samples. Estimated human blood indices ranged from 53%-76% for Ae. aegypti, 32%-82% for Ae. simpsoni s.l. and 26%-61% for Cx. pipiens s.l. and were consistently lower for Sanger-based sequencing approach compared to Miseq-based sequencing approach. These findings demonstrate that Miseq sequencing approach is superior to Sanger sequencing approach as it can reliably identify mixed host blood meals in a single mosquito, improving our ability to understand the transmission dynamics of mosquito-borne pathogens.

Despite the well documented link between cover cropping and soil microbiology, the influence of specific cover crop species on soil microbes remains poorly understood. We evaluated how soil fungal communities in a no till system respond to four cover crop treatments: no cover crop (REF), cereal ryegrass (CRYE), wild pennycress (WPEN), and a mix of pea, clover, radish, and oat (PCRO). Soil samples were collected from experimental plots following termination of cover crops from depths of 0-2 cm and 2-4 cm where cover crops had significantly increased soil organic matter. There was no significant interaction between soil depth and cover crop treatment on either alpha diversity or beta diversity. All cover crop treatments (CRYE, PCRO, and WPEN) enhanced soil fungal richness but only CRYE enhanced soil fungal diversity and altered the fungal community structure. Soil depth altered the fungal community structure but had no effect on fungal diversity and richness. Genus Fusarium which includes some of the most economically destructive pathogens was more abundant in REF and PCRO treatments compared to CRYE and WPEN. In contrast, genus Mortierella which is known to promote plant health was more abundant in all cover crop treatments relative to the REF. These findings demonstrate that cover cropping can increase soil fungal species richness and alter fungal community structure, potentially promoting the abundance of beneficial fungi and reducing the abundance of some plant pathogens within the genus Fusarium. These effects are dependent on cover crop species, a factor that should be considered when selecting appropriate cover crops for a particular cropping system.

The assembly of host-associated microbial communities is influenced by multiple factors, but the effect of microbiomes on host phenotypes is often not well understood. To address questions of food-web effects on host microbiome assembly, we manipulated the resource environment (grass only [G] vs. grass + nutrients [GN]), competition type (intra- vs. inter-specific) and density (high vs. low) for Culex restuans mosquito larvae. We predicted the microbial communities in fourth-instar larvae would differ between these environmental treatments and that these treatments would translate into differences in the adult phenotype. Resource environment and density influenced the larval microbiome. In addition, the larval microbiome exhibited notable differences compared to the free-living microbial communities. Resource-driven differences in the larval samples can be attributed to Arcobacteraceae being more abundant in larvae reared in the GN treatments relative to those reared in the G treatments and Comamonadaceae being more abundant in the G treatment. Although significant, the difference in community structure between density treatments was difficult to discern. This appears to be driven by Weeksellaceae only being abundant in the high-density, interspecific, GN treatment. Rearing larvae to adulthood under severe food limitation resulted in low survival (<25%) in both resource environments. Approximately 60% of survivors to adulthood were male. Larvae reared in the intraspecific, G treatment had the shortest development time to adulthood and emerged as the smallest adults. These results demonstrate how environmental variation can significantly alter the alpha and beta diversity of free-living microbes, which in turn can significantly affect host phenotype and critical life history traits, such as development time, size at adulthood, and survival. These findings highlight the importance of considering environmental influences on microbiome diversity to understand and predict host outcomes, offering valuable insights for diverse applications in fields such as ecology, public health, and agriculture.

Dormant propagules can provide a rapid colonization source for temporary aquatic habitats and set the trajectory for community dynamics, yet the egg banks of stormwater management systems have received little attention. We asked which species hatched from the sediment of drainage ditches in Champaign County, IL, and found bdelloid rotifers and ostracods (Heterocypris incongruens) to be the most common taxa. These sites also are colonized by mosquitoes, and we established laboratory experiments to examine interspecific interactions between common co-occurring taxa. Culex restuans larvae were reared in the presence or absence of H. incongruens at two intra- and interspecific densities (20 or 40 total individuals) and their survivorship to adulthood, development time to adulthood, adult body size, and sex ratio were determined. Survival for Cx. restuans was significantly lower at high larval density than at low larval density in both treatments. Culex restuans larvae reared in the presence of H. incongruens had a shorter development time to adulthood and emerged as larger adults compared to those reared in the absence of H. incongruens. The sex ratios in the H. incongruens treatments were female-biased whereas those in the Culex-only treatments were male-biased. These differences may have epidemiological implications, as only female mosquitoes serve as disease vectors. Our results emphasize the importance of understanding interspecific interactions in influencing larval mosquito development traits.

The cabbage looper Trichoplusia ni is an important agricultural pest worldwide and is frequently used as a model organism for assessing entomopathogenic fungi virulence, though few studies have measured the host response repertoire to fungal biocontrol agents. Here, we quantified the immune response of T. ni larvae following exposure to two entomopathogenic fungal species: Beauveria bassiana and Cordyceps javanica. Results from our study demonstrate that T. ni larvae exposed to fungal entomopathogens had higher total phenoloxidase activity compared to controls, indicating that the melanization cascade is one of the main immune components driving defense against fungal infection and contrasting observations from other insect-fungi interaction studies. We also observed differences in host response depending on the species of entomopathogenic fungi, with significantly higher induction observed during infections with B. bassiana than with C. javanica. Larvae exposed to B. bassiana had an increased expression of genes involved in prophenoloxidase response and the Imd, JNK, and Jak/STAT immune signaling pathways. Our results indicate a notable absence of Toll pathway-related responses, further contrasting results to other insect-fungi pathosystems. Important differences were also observed in the induction of antimicrobial effectors, with B. bassiana infections eliciting three antimicrobial effectors (lysozyme, gloverin, and cecropin), while C. javanica only induced cecropin expression. These results provide insight into the host response strategies employed by T. ni for protection against entomopathogenic fungi and increase our understanding of insect-fungal entomopathogen interactions, aiding in the design of more effective microbial control strategies for this important agricultural pest.

A general method to spin-label a fatty acid was demonstrated as well as an assay of the effectiveness of methyl 10-undecenoate and the spin-labeled version, against the larvae of Aedes aegypti. The LC50s were 66 and 58 μL/120 mL (55 and 48 ppm) respectively, and the LC90s were 108 and 90 μL/120 mL (113 and 90) ppm. This shows that the spin-label has very little effect on the larvicidal activity of the compound. This opens the possibility of the use of spin-labeling as a tool to determine mechanisms of larvicidal effectiveness, as it can be employed without altering the system under study.

Strategic, sustainable, and ecofriendly alternatives to chemical pesticides are needed to effectively control mosquitoes and reduce the incidence of their vectored diseases. We evaluated several Brassicaceae (mustard family) seed meals as sources of plant derived isothiocyanates produced from the enzymatic hydrolysis of biologically inactive glucosinolates for the control of Aedes aegypti (L., 1762). Five defatted seed meals (Brassica juncea (L) Czern., 1859, Lepidium sativum L., 1753, Sinapis alba L., 1753, Thlaspi arvense L., 1753, and Thlaspi arvense-heat inactivated and three major chemical products of enzymatic degradation (allyl isothiocyanate, benzyl isothiocyanate and 4-hydroxybenzyl isothiocyanate) were assayed to determine toxicity (LC50) to Ae. aegypti larvae. All seed meals except the heat inactivated T. arvense were toxic to mosquito larvae. L. sativum seed meal was the most toxic treatment to larvae (LC50 = 0.04 g/120 mL dH2O) at the 24-h exposure. At the 72-h evaluation, the LC50 values for B. juncea, S. alba and T. arvense seed meals were 0.05, 0.08 and 0.1 g/120 mL dH2O, respectively. Synthetic benzyl isothiocyanate was more toxic to larvae 24-h post treatment (LC50 = 5.29 ppm) compared with allyl isothiocyanate (LC50 = 19.35 ppm) and 4-hydroxybenzyl isothiocyanate (LC50 = 55.41 ppm). These results were consistent with the higher performance of the benzyl isothiocyanate producing L. sativum seed meal. Isothiocyanates produced from seed meals were more effective than the pure chemical compounds, based on calculated LC50 rates. Using seed meal may provide an effective method of delivery for mosquito control. This is the first report evaluating the efficacy of five Brassicaceae seed meals and their major chemical constituent against mosquito larvae and demonstrates how natural compounds from Brassicaceae seed meals can serve as a promising ecofriendly larvicides to control mosquitoes.

Aedes aegypti transmits major arboviruses of public health importance, including dengue, chikungunya, Zika, and yellow fever. The use of insecticides represents the cornerstone of vector control; however, insecticide resistance in Ae. aegypti has become widespread. Understanding the molecular basis of insecticide resistance in this species is crucial to design effective resistance management strategies. Here, we applied Illumina RNA-Seq to study the gene expression patterns associated with resistance to three widely used insecticides (malathion, alphacypermethrin, and lambda-cyhalothrin) in Ae. aegypti populations from two sites (Manatí and Isabela) in Puerto Rico (PR). Cytochrome P450s were the most overexpressed detoxification genes across all resistant phenotypes. Some detoxification genes (CYP6Z7, CYP28A5, CYP9J2, CYP6Z6, CYP6BB2, CYP6M9, and two CYP9F2 orthologs) were commonly overexpressed in mosquitoes that survived exposure to all three insecticides (independent of geographical origin) while others including CYP6BY1 (malathion), GSTD1 (alpha-cypermethrin), CYP4H29 and GSTE6 (lambda-cyhalothrin) were uniquely overexpressed in mosquitoes that survived exposure to specific insecticides. The gene ontology (GO) terms associated with monooxygenase, iron binding, and passive transmembrane transporter activities were significantly enriched in four out of six resistant vs. susceptible comparisons while serine protease activity was elevated in all insecticide-resistant groups relative to the susceptible strain. Interestingly, cuticular-related protein genes (chinase and chitin) were predominantly downregulated, which was also confirmed in the functional enrichment analysis. This RNA-Seq analysis presents a detailed picture of the candidate detoxification genes and other pathways that are potentially associated with pyrethroid and organophosphate resistance in Ae. aegypti populations from PR. These results could inform development of novel molecular tools for detection of resistance-associated gene expression in this important arbovirus vector and guide the design and implementation of resistance management strategies.

Beneficial microorganisms and plant essential oils are key components of sustainable agriculture, but knowledge of their compatibility is limited. We investigated the effect of 23 essential oils on three beneficial bacterial species ( Bacillus cereus , Bacillus velezensis and Priestia megaterium ) that are commercially used as biostimulants and biocontrol antagonists. Essential oils were tested at a dilution of 1:10, the highest economical use rate anticipated in crop protection applications. Seventeen of the 23 essential oils had no inhibitory activity against any of the three bacterial species. Onion essential oil had strong activity against B. velezensis and no activity against B. cereus or P. megaterium . Palmarosa and pimenta essential oils were active against B. velezensis and inactive against B. cereus and P. megaterium . Coriander essential oil was inactive against B. velezensis , active against B. cereus , and strongly active against P. megaterium. Oregano essential oil was active against the three bacterial species while garlic essential oil was active against B. cereus and strongly active against B. velezensis and P. megaterium . Allyl disulfide, carvacrol, eugenol, and geraniol, the major constituents of garlic, oregano, pimenta leaf, and palmarosa/coriander essential oils, respectively, were active or strongly active against at least two of the three bacterial species suggesting their contribution to the antimicrobial activity of these essential oils. These findings indicate that most of the essential oils tested are compatible with beneficial microbes and can easily be integrated with beneficial microbes in organic cropping systems. Further studies should investigate how to harness the pesticidal properties of the six active essential oils while limiting their detrimental effect on beneficial microbes.

Mosquito resistance to microbial infections, including fungal entomopathogens that are selected for mosquito control, depend on a range of antimicrobial effectors, among them antimicrobial peptides (AMPs). These short peptides, along the antimicrobial effector lysozyme, act by disrupting the microbial cell membrane or by interfering with microbial physiological processes. While the induction of AMPs and lysozyme during fungal entomopathogenic infections have been reported, their contribution to the mosquito antifungal response has not been evaluated. In this study, we assessed the induction of Ae. aegypti AMPs and lysozyme genes at two points of infection and against distinct entomopathogenic fungi. Our results indicate that fungal infection elicits the expression of cecropin, defensin, diptericin, holotricin, and lysozyme, but do not affect those of attacin or gambicin. We further evaluated the role of these antimicrobial effectors via RNAi-based depletion of select AMPs during challenges with two entomopathogenic fungi. Our results reveal that AMPs and lysozyme are critical to the antifungal response, acting in concert, rather than individually, to potentiate their antimicrobial effect against entomopathogenic fungi. This study further contributes to a better understanding of the mechanisms that confer resistance to entomopathogenic fungi in an important mosquito vector.

The repellency and toxicity of a CO 2 -derived cedarwood oil (CWO) was evaluated against actively questing unfed nymphs of four species of hard ticks: Amblyomma americanum (L.), Dermacentor variabilis (Say), Ixodes scapularis Say, and Rhipicephalus sanguineus (Latreille). Using a vertical climb bioassay for repellency, nymphs of these species avoided a CWO-treated filter paper in proportional responses to treatment concentrations. At 60 min of exposure, I. scapularis nymphs were most sensitive with 50% repellency concentration (RC 50 ) of 19.8 µg cm −2 , compared with RC 50 of 30.8, 83.8 and 89.6 µg cm −2 for R. sanguineus, D. variabilis and A. americanum , respectively. Bioassays determined the lethal concentration for 50% (LC 50 ) and 90% (LC 90 ) mortality of nymphs exposed to CWO in treated vials after 24- and 48-h exposure. After 24 h exposure, the LC 50 values were 1.25, 3.45 and 1.42 µg cm −2 and LC 90 values were 2.39, 7.59 and 4.14 µg cm −2 for D. variabilis , I. scapularis and R. sanguineus , respectively, but had minimal effect on A. americanum . After 48 h exposure, the LC 50 values were 4.14, 0.78, 0.79 and 0.52 µg cm −2 , and LC 90 values were 8.06, 1.48, 1.54 and 1.22 µg cm −2 for A. americanum , D. variabilis , I. scapularis and R. sanguineus , respectively. The repellency of CWO on tick species decreased with time. The repellency and toxicity bioassays demonstrated concentration-dependent responses of tick nymphs to the oil, indicating the potential of the CO 2 -derived cedarwood oil be developed as an eco-friendly repellent and/or acaricide.

Background The guts of blood-sucking insects host a community of bacteria that can shift dramatically in response to biotic and abiotic factors. Identifying the key factors structuring these microbial communities has important ecological and epidemiological implications. Methods We used the yellow fever mosquito, Aedes aegypti , to investigate the impact of mixed blood meals on gut microbiota of vector mosquitoes. Adult females were experimentally fed on sugar or blood from chicken, rabbit or a mixture of chicken and rabbit blood, and their gut microbiota were characterized using 16S rRNA gene amplification and MiSeq sequencing. Results The gut bacterial communities of mosquitoes fed on the three blood meal treatments clustered separately, suggesting that host species identity and mixed blood-feeding are key determinants of gut bacterial community composition in mosquitoes. Mixed blood meal had a synergistic effect on both operational taxonomic unit (OTU) richness and the Shannon diversity index, suggesting that mixed blood-feeding can offset the nutritional deficit of blood meals from certain host species. The microbial communities observed in this study were distinct from those identified from similarly fed Ae. aegypti from our previous study. Conclusions These findings demonstrate that vector host-feeding preferences can influence gut microbial composition and diversity, which could potentially impact pathogen acquisition and transmission by the vector. The results also demonstrate that different microenvironmental conditions within the laboratory may play an important role in structuring the microbial communities of independently reared mosquito colonies. Graphical Abstract

Mosquito bacterial communities are essential in mosquito biology, and knowing the factors shaping these bacterial communities is critical to their application in mosquito-borne disease control. This study investigated how the larval environment influences the bacterial communities of larval stages of two container-dwelling mosquito species, Aedes triseriatus, and Aedes japonicus. Larval and water samples were collected from tree holes and used tires at two study sites, and their bacteria characterized through MiSeq sequencing of the 16S rRNA gene. Bacterial richness was highest in Ae. japonicus , intermediate in Ae. triseriatus , and lowest in water samples. Dysgonomonas was the dominant bacterial taxa in Ae. triseriatus larvae; the unclassified Comamonadaceae was dominant in water samples from waste tires, while Mycobacterium and Carnobacterium , dominated Ae. japonicus . The two mosquito species harbored distinct bacterial communities that were different from those of the water samples. The bacterial communities also clustered by habitat type (used tires vs. tree holes) and study site. These findings demonstrate that host species, and the larval sampling environment are important determinants of a significant component of bacterial community composition and diversity in mosquito larvae and that the mosquito body may select for microbes that are generally rare in the larval environment.

Insecticide resistance has emerged as a persistent threat to the fight against vector-borne diseases. We compared the gut microbiota of permethrin-selected (PS) strain of Aedes aegypti relative to the parent (KW) strain from Key West, Florida. Bacterial richness but not diversity was significantly higher in PS strain compared to KW strain. The two mosquito strains also differed in their gut microbial composition. Cutibacterium spp., Corynebacterium spp., Citricoccus spp., Leucobacter spp., Acinetobacter spp., Dietzia spp., and Anaerococcus spp. were more abundant in PS strain than in KW strain. In contrast, Sphingomonas spp., Aquabacterium spp., Methylobacterium spp., Flavobacterium spp., Lactobacillus spp., unclassified Burkholderiaceae and unclassified Nostocaceae were more abundant in KW strain compared to PS strain. PS strain was enriched with propionate metabolizers, selenate reducers, and xylan, chitin, and chlorophenol degraders while KW strain was enriched with sulfur oxidizers, sulfur metabolizers, sulfate reducers and naphthalene and aromatic hydrocarbons degraders. These findings demonstrate an association between the gut microbiota and insecticide resistance in an important vector species and sets the foundation for future studies to investigate the contribution of gut microbiota to evolution of insecticide resistance in disease vectors.

BACKGROUND:The rapid and widespread evolution of insecticide resistance has emerged as one of the major challenges facing malaria control programs in sub-Saharan Africa. Understanding the insecticide resistance status of mosquito populations and the underlying mechanisms of insecticide resistance can inform the development of effective and site-specific strategies for resistance prevention and management. The aim of this study was to investigate the insecticide resistance status of Anopheles gambiae (s.l.) mosquitoes from coastal Kenya. METHODS:Anopheles gambiae (s.l.) larvae sampled from eight study sites were reared to adulthood in the insectary, and 3- to 5-day-old non-blood-fed females were tested for susceptibility to permethrin, deltamethrin, dichlorodiphenyltrichloroethane (DDT), fenitrothion and bendiocarb using the standard World Health Organization protocol. PCR amplification of rDNA intergenic spacers was used to identify sibling species of the An. gambiae complex. The An. gambiae (s.l.) females were further genotyped for the presence of the L1014S and L1014F knockdown resistance (kdr) mutations by real-time PCR. RESULTS:Anopheles arabiensis was the dominant species, accounting for 95.2% of the total collection, followed by An. gambiae (s.s.), accounting for 4.8%. Anopheles gambiae (s.l.) mosquitoes were resistant to deltamethrin, permethrin and fenitrothion but not to bendiocarb and DDT. The L1014S kdr point mutation was detected only in An. gambiae (s.s.), at a low allelic frequency of 3.33%, and the 1014F kdr mutation was not detected in either An. gambiae (s.s.) or An. arabiensis. CONCLUSION:The findings of this study demonstrate phenotypic resistance to pyrethroids and organophosphates and a low level of the L1014S kdr point mutation that may partly be responsible for resistance to pyrethroids. This knowledge may inform the development of insecticide resistance management strategies along the Kenyan Coast.