Face à la résistance croissante de Anopheles gambiae s.l. aux pyréthrinoïdes, des approches complémentaires aux moustiquaires imprégnées d’insecticide sont nécessaires. Cette étude a évalué, en cases expérimentales à la Vallée du Kou (Burkina Faso), l’efficacité des appâts sucrés toxiques attractifs (ASTA) à base de Bacillus thuringiensis israelensis (Bti), dénommés Bti Sugar Patches (BSP), combinés à une moustiquaire non imprégnée ou imprégnée à la deltaméthrine. Deux protocoles ont été conduits : lâcher-recapture et vol libre. Les indicateurs mesurés étaient l’exophilie, le gorgement et la mortalité. En lâcher-recapture, la moustiquaire imprégnée + 400 BSP a donné la plus faible proportion de femelles gorgées (14% contre 68% pour le contrôle) et la plus forte inhibition du repas de sang (80%). La mortalité la plus élevée a été observée avec la moustiquaire imprégnée + 400 BSP (36%). En vol libre, la mortalité maximale a été obtenue avec la moustiquaire non imprégnée + 400 BSP (24%), contre 8% avec la moustiquaire imprégnée + 400 BSP. Ces résultats suggèrent que les BSP peuvent réduire le contact homme-vecteur et induire une mortalité additionnelle, mais que l’efficacité en vol libre reste à optimiser. ENGLISH ABSTRACT With increasing pyrethroid resistance in Anopheles gambiae s.l., complementary approaches to insecticide-treated nets are needed. We evaluated, in experimental huts in the Vallée du Kou (Burkina Faso), the efficacy of attractive toxic sugar baits (ATSBs) based on Bacillus thuringiensis israelensis (Bti), referred to as Bti Sugar Patches (BSP), used in combination with either an untreated net or a deltamethrin-treated net. Two protocols were conducted: release-recapture and free-flight. Outcomes included exophily, blood-feeding and mortality. In release-recapture, the deltamethrin-treated net + 400 BSP yielded the lowest blood-feeding rate (14% vs 68% in the untreated control) and the highest blood-feeding inhibition (80%). The highest mortality was observed with the treated net + 400 BSP (36%). In free-flight, mortality was highest with the untreated net + 400 BSP (24%), compared with 8% for the treated net + 400 BSP. These findings suggest that BSP may reduce human-vector contact and add mortality, although efficacy under free-flight conditions should be optimized. Keywords: attractive toxic sugar baits, Bacillus thuringiensis israelensis, Anopheles gambiae s.l., experimental huts, pyrethroid resistance, insecticide-treated nets.

BackgroundAedes aegypti vectors several important arboviruses including dengue and yellow fever. This vector mosquito is controlled mainly by using synthetic insecticides and repellents. Overusing these insecticides causes mosquito resistance, harms the environment, and affects human health. This report reevaluates the repellent activities of Cymbopogon nardus, Eucalyptus camaldulensis essential oils (EOs), and their mixtures against laboratory-reared adult Ae. aegypti.MethodsThe chemical composition of C. nardus, E. camaldulensis EOs, and their 1:1 combination was identified by gas chromatography-mass spectrometry (GC-MS). We evaluated the repellent activities of these oils against Ae. aegypti using a Y-maze olfactometer. The preference index (PI) was evaluated and compared with the binary data obtained from the olfactometer assay with samples that did not contain EOs (control) using an Exact Binomial test (α= 0.05)ResultsSeveral monoterpenes and sesquiterpene compounds were found in EOs and their mixture. The EOs of E. camaldulensis and the mixture of the two oils showed a repellent activity of 50%, whereas C. nardus was less active and attracted mosquitoes at 1 ppm.ConclusionWe show that EOs from C. nardus and E. camaldulensis contain compounds that repel Ae. aegypti. Future studies will identify specific compounds with the highest repellent activities and use them to formulate in the future a potent repellent against Ae. aegypti for human protection.

Plant-derived insecticides, such as essential oils, can be an effective alternative to replace synthetic chemical insecticides against Spodoptera frugiperda, which becomes increasingly resistant to synthetic products. This study aims to evaluate essential oils (EOs) effects on larval growth and development following feeding inhibition, growth regulation, and repellency of EOs of Lippia multiflora (Verbenaceae), Cymbopogon schoenanthus (Poaceae), and their combination. Topical application of EOs was used on S. frugiperda larvae for larvicidal effect or treating filter paper with the EOs to find repellency. The effect of EOs on food intake and larval growth was also evaluated. Several types of compounds have been identified in the EOs, mainly monoterpenes with the appearance of new compounds in the 1 : 1 combination. Bioassay results show that individuals and combinations of EOs significantly influenced S. frugiperda larval development. L. multiflora caused 100% mortality of L2 larvae within 24 hours at 3%. The Lm + Cs (1 : 1) EOs combination was the most effective with LC50 and LC90 of 1.02% and 1.92%, respectively. Lm + Cs (1/4 : 3/4) EOs caused the highest inhibition of food consumption, 0.0160 g consumed after food was treated with 2.2% concentration compared to food consumption of 0.0602 g for the control group at 24 hours. Lower food consumption caused the inhibition of larval growth and weight loss of 0.0005 g/day at the 2.2% EOs concentration. The highest repellency effect of the EOs was found in EOs of L. multiflora, exhibiting a repulsion of 83.33% of the larvae after 3 hours of exposure. This diversity in the biological actions of the EOs tested on S. frugiperda represents valuable options for contributing to integrated pest management and an alternative to synthetic chemical insecticides.

The massive and inappropriate use of synthetic insecticides is causing significant and increasing environmental disruption. Therefore, developing effective natural mosquitocidal compounds could be an alternative tool for malarial vector control. The present study investigates the larvicidal and adulticidal effect of methanol and acetone extracts of leaves from Lippia chevalieri, Lippia multiflora, Cymbopogon schoenanthus, and Lantana camara against Anopheles arabiensis, to control the most widespread vector transmitting malaria in sub-Saharan. Africa. Extracts were evaluated following WHO modified test procedure against third- to fourth-instar larvae and, non-blood-fed females from 3- to 5-day-old field populations of An. arabiensis under laboratory conditions using WHO larval and CDC bottle bioassays, respectively. Mortality was recorded after 24-h exposure and several compounds were identified in the extracts. The methanolic and acetonic extracts of L. camara were effective against larvae showing lethal concentrations to 50% (LC50) of the population, at 89.48 and 58.72 ppm, respectively. The acetonic extracts of C. schoenanthus and L. chevalieri showed higher toxicities LC50s of 0.16% and 0.22% against female adults, respectively. The methanolic extracts of L. multiflora and L. chevalieri LC50s were effective at 0.17% and 0.27%, respectively, against female adults. These results indicate that the plant extracts tested may represent effective means to control An. arabiensis when used to treat the surface of the marshes.

Heliothis virescens larval chymotrypsin (GenBank accession number AF43709) was cloned, sequenced and its three dimensional (3D) conformation modeled. The enzyme's transcript was first detected 6 days after larval emergence and the transcript level was shown to fall between larval ecdysis periods. Comparisons between the activities of larval gut chymotrypsin and trypsin shows that chymotrypsin activity is only 16% of the total trypsin activity and the pH optimum of the larval chymotrypsin is between pH 9-10, however the enzyme also exhibited a broad activity between pH 4-6. Injections of AeaTMOF and several shorter analogues into 3rd instar larvae followed by Northern blot analyses showed that although the chymotrypsins activities were inhibited by 60%-80% the transcript level of the sequenced chymotrypsin was not reduced and was similar to controls in which the chymotrypsin activity was not inhibited, indicating that AeaTMOF and its analogues exert a translational control. Based on these observations a putative AeaTMOF receptor (ABCC4) homologous to the Ae. aegypti ABC receptor sequence was found in the H. virescens genome. 3D molecular modeling and docking of the AeaTMOF and several of its analogues to the ABCC4 receptor showed that it can bind AeaTMOF and its analogues as was shown before for the Ae. aegypti receptor.

Aedes aegypti adult and larval blood downregulated chymotrypsin II was cloned, sequenced and its 3D conformation modeled. Cloning of the enzymes from adult and larval guts indicated that both genes sit at the same location on Chromosome 2. Genomic analyses showed that larval and adult genes are the same and both have four exons and three introns that are located on an 8.32 Kb DNA in direction with the Ae. aegypti genome. The adult and larval transcript synthesis is controlled by alternative splicing explaining small difference in the amino acids sequences. Chymotrypsin II that was extracted from guts of sugar-fed and at 48 after blood feeding showed a pH optimum of 4-5 with a broad shoulder of activity from pH 6 to 10. Dot blot analyses show that the enzyme's transcript is downregulated after females take a blood meal and upregulated at 48 h after the blood meal. A Chymotrypsin II transcript was also detected in the larval gut during different times of larval developmental stages, indication that Ae. aegypti chymotrypsin II is synthesized by adults and larval guts. The possibility that JH III and 20HE play an active role in the regulation is discussed.

The Fall Armyworm (FAW), Spodoptera frugiperda is one of the main crop pests in Burkina Faso.The growing resistance of FAW to chemical insecticides and their harmful effects on humans and the environment necessitates the search for biodegradable and eco-friendly substances.Therefore, alternative control methods, such as the use of essential oils (EOs), are important in finding new strategies for the effective management of this pest.This study aims to evaluate the biological properties of EOs extracted from leaves of C. schoenanthus, L. multiflora, and O. americanum on larvae and pupae of S. frugiperda.The activities of these oils were evaluated by topical treatment of different concentrations on L2 larvae and pupae and their effect on food intake.Topical application to L2 caused dose dependent mortality and EO of O. americanum was most effective at 72h with an LC50 of 0.3%.Application of L. multiflora EO caused mortality with a LC50 of 0.4%.EO from C. schoenanthus caused LC50 and LC90 of 0.8% and 12.5%, respectively.The oils of L. multiflora and C. schoenanthus have been shown to be most effective against pupae.L. multiflora and C. schoenanthus EOs showed significant effects within 24 h after food intake.The EOs caused inhibition of larval growth and a weight loss of 0.002% per day using 1% L. multiflora EOs and 0.003% using 2% C. schoenanthus EOs.The inhibitory effects of these oils could be exploited using integrated pest management for S. frugiperda.

Rapid development of resistance in vector mosquitoes to synthetic insecticides is a major challenge for malaria control. The use of plant-derived essential oils (EOs) is an attractive strategy in controlling mosquito populations because they are environmentally safe and may have a lower chance of developing resistance. This study assessed the larvicidal activities of EOs from Lantana camara, Lippia multiflora, Lippia chevalieri, and Cymbopogon schoenanthus against Anopheles funestus and Culex quinquefasciatus. The 3rd–4th instar larvae were tested using a World Health Organization (WHO)-modified protocol to evaluate larval mortality 24 h after exposure to EOs and their binary combinations. Culex quinquefasciatus larvae were more susceptible to EOs than An. funestus larvae. For Cx. quinquefasciatus, the lethal concentrations at 50% mortality (LC50s) of EOs from C. schoenanthus, L. multiflora, L. camara, and L. chevalieri were 23.32, 27.24, 38.54, and 54.11 ppm, respectively; whereas for An. funestus, the EO LC50s were 120.5, 67.5, 49.21, and 105.74 ppm, respectively. Synergistic effects were observed using EOs from C. schoenanthus + L. multiflora (LC50 = 44.05 ppm) on An. funestus, while L. camara + L. chevalieri (LC50 = 33.16 ppm), L. chevalieri + C. schoenanthus (LC50 = 12.08 ppm), and L. multiflora + L. chevalieri (LC50 = 20.61 ppm) were synergistic for Cx. quinquefasciatus. These results indicate the potential of EOs derived from local plants and their binary combinations as botanical larvicides. The EOs could be used as future ecofriendly agents to control these vectors.

A Biomolecules Special Issue on insect receptors was a great opportunity to invite colleagues from all over the world to contribute original articles and timely reviews on the subject [...]

Botanical biopesticides have potential for use in mosquito control because they exhibit low mammalian toxicity, are readily biodegraded, show target specificity and insecticidal activity. As populations of mosquito species grow more resistant to currently used organic insecticides, a need for new and effective insecticides for vector control becomes more urgent. This study reports the effects of synergistic and antagonistic essential oils (EOs) from Cymbopogon schoenanthus, Lantana camara, Lippia chevalieri and Lippia multiflora and their binary combinations against Anopheles gambiae s. l. larvae and adults. EOs insecticidal properties were tested with third to fourth-instar larvae and, non-blood-fed 3-5-day old field-collected An. gambiae using WHO and CDC bottle bioassays, respectively. Many compounds were found in the EOs mixtures. All EOs showed larvicidal and adulticidal activities with mortality from 0 to 100% against An. gambiae which were concentration dependent and EOs specific. EO mixture from C. schoenanthus and L. multiflora showed synergistic effect with LC50 of 38 ppm and EOs from L. chevalieri and C. schoenanthus showed antagonistic effect against larvae with LC50 of 100.84 ppm. On the other hand, EOs combinations showed additive effect on all adults. This study describes the potential of using EOs and their synergistic mixtures as potential insecticide(s) as a safer alternative to synthetic insecticides.

Juvenile hormone epoxide hydrolase (JHEH) plays an important role in the metabolism of JH III in insects. To study the control of JHEH in female Drosophila melanogaster, JHEH 1, 2 and 3 cDNAs were cloned and sequenced. Northern blot analyses showed that the three transcripts are expressed in the head thorax, the gut, the ovaries and the fat body of females. Molecular modeling shows that the enzyme is a homodimer that binds juvenile hormone III acid (JH IIIA) at the catalytic groove better than JH III. Analyses of the three JHEH promoters and expressing short promoter sequences behind a reporter gene (lacZ) in D. melanogaster cell culture identified a JHEH 3 promoter sequence (626 bp) that is 10- and 25-fold more active than the most active promoter sequences of JHEH 2 and JHEH 1, respectively. A transcription factor (TF) Sp1 that is involved in the activation of JHEH 3 promoter sequence was identified. Knocking down Sp1 using dsRNA inhibited the transcriptional activity of this promoter in transfected D. melanogaster cells and JH III and 20HE downregulated the JHEH 3 promoter. On the other hand, JH IIIA and farnesoic acid did not affect the promoter, indicating that JH IIIA is JHEH’s preferred substrate. A transgenic D. melanogaster expressing a highly activated JHEH 3 promoter behind a lacZ reporter gene showed promoter transcriptional activity in many D. melanogaster tissues.

The antimicrobial properties of proline-rich Aedes aegypti decapeptide TMOF (AeaTMOF) and oncocin112 (1–13) were compared. Incubations with multidrug-resistant Escherichia coli cells showed that AeaTMOF (5 mM) was able to completely inhibit bacterial cell growth, whereas oncocin112 (1–13) (20 mM) partially inhibited bacterial growth as compared with bacterial cells that were not multidrug-resistant cells. AeaTMOF (5 mM) was very effective against Acinetobacter baumannii and Pseudomonas aeruginosa, completely inhibiting cell growth during 15 h incubations. AeaTMOF (5 mM) completely inhibited the Gram-positive bacteria Staphylococcus aureus and Bacillus thurengiensis sups. Israelensis cell growth, whereas oncocin112 (1–13) (10 and 20 mM) failed to affect bacterial cell growth. E. coli cells that lack the SbmA transporter were inhibited by AeaTMOF (5 mM) and not by oncocin112 (1–13) (10 to 20 mM), indicating that AeaTMOF can use other bacterial transporters than SbmA that is mainly used by proline-rich antimicrobial peptides. Incubation of E. coli cells with NaAzide showed that AeaTMOF does not use ABC-like transporters that use ATP hydrolysis to import molecules into bacterial cells. Three-dimensional modeling and docking of AeaTMOF to SbmA and MdtM transporters showed that AeaTMOF can bind these proteins, and the binding location of AeaTMOF inside these protein transporters allows AeaTMOF to be transported into the bacterial cytosol. These results show that AeaTMOF can be used as a future antibacterial agent against both multidrug-resistant Gram-positive and -negative bacteria.

Juvenile hormone epoxide hydrolase (JHEH) plays an important role in the metabolism of juvenile hormone III (JH III) in insects. To study the role that JHEH plays in female Aedes aegypti JHEH 1, 2, and 3 complementary DNA (cDNAs) were cloned and sequenced. Northern blot analyses show that the three transcripts are expressed in the head thorax, the gut, the ovaries, and the fat body of females. Molecular modeling shows that the enzyme is a homodimer that binds JH III acid (JH IIIA) at the catalytic groove better than JH III. The cDNA of JHEH 1 and 2 are very similar indicating close relationship. Knocking down of jheh 1, 2, and 3 in adult female and larval Ae. aegypti using double-stranded RNA (dsRNA) did not affect egg development or caused adult mortality. Larvae that were fed bacterial cells expressing dsRNA against jheh 1, 2, and 3 grew normally. Treating blood-fed female Ae. aegypti with [12-3 H](10R) JH III and analyzing the metabolites by C18 reversed phase chromatography showed that JHEH preferred substrate is not JH III but JH IIIA. Genomic analysis of jheh 1, 2, and 3 indicate that jheh 1 and 2 are transcribed from a 1.53 kb DNA whereas jheh 3 is transcribed from a 10.9 kb DNA. All three genes are found on chromosome two at distinct locations. JHEH 2 was expressed in bacterial cells and purified by Ni affinity chromatography. Sequencing of the recombinant protein by MS/MS identified JHEH 2 as the expressed recombinant protein.

Aedes aegypti Trypsin Modulating Oostatic Factor (AeaTMOF). a mosquito decapeptide that controls trypsin biosynthesis in female and larval mosquitoes. enters the gut epithelial cells of female mosquitoes using ABC-tmfA receptor/importer. To study the ultimate targeted receptor after AeaTMOF enters the cell, AeaTMOF was incubated in vitro with either Escherichia coli or Spodoptera frugiperda protein-expressing extracts containing 70S and 80S ribosomes, respectively. The effect of AeaTMOF on luciferase biosynthesis in vitro using 70S ribosomes was compared with that of oncocin112 (1–13), a ribosome-binding antibacterial peptide. The IC50 of 1 μM and 2 μM, respectively, for both peptides was determined. Incubation with a protein-expressing system and S. frugiperda 80S ribosomes determined an IC50 of 1.8 μM for Aedes aegypti larval late trypsin biosynthesis. Incubation of purified E. coli ribosome with increasing concentration of AeaTMOF shows that the binding of AeaTMOF to the bacterial ribosome exhibits a high affinity (KD = 23 ± 3.4 nM, Bmax = 0.553 ± 0.023 pmol/μg ribosome and Kassoc = 4.3 × 107 M−1). Molecular modeling and docking experiments show that AeaTMOF binds bacterial and Drosophila ribosome (50S and 60S, respectively) at the entrance of the ribosome exit tunnel, blocking the tRNA entrance and preventing protein biosynthesis. Recombinant E. coli cells that express only ABC-tmfA importer are inhibited by AeaTMOF but not by oncocin112 (1–13). These results suggest that the ribosome is the ultimate targeted receptor of AeaTMOF.

EDITORIAL article Front. Physiol., 09 December 2021Sec. Invertebrate Physiology https://doi.org/10.3389/fphys.2021.815010

Trypsin Modulating Oostatic Factor (TMOF) receptor was solubilized from the guts of female Ae. Aegypti and cross linked to His6-TMOF and purified by Ni affinity chromatography. SDS PAGE identified two protein bands (45 and 61 kDa). The bands were cut digested and analyzed using MS/MS identifying a protein sequence (1306 amino acids) in the genome of Ae. aegypti. The mRNA of the receptor was extracted, the cDNA sequenced and cloned into pTAC-MAT-2. E. coli SbmA− was transformed with the recombinant plasmid and the receptor was expressed in the inner membrane of the bacterial cell. The binding kinetics of TMOF-FITC was then followed showing that the cloned receptor exhibits high affinity to TMOF (KD = 113.7 ± 18 nM ± SEM and Bmax = 28.7 ± 1.8 pmol ± SEM). Incubation of TMOF-FITC with E. coli cells that express the receptor show that the receptor binds TMOF and imports it into the bacterial cells, indicating that in mosquitoes the receptor imports TMOF into the gut epithelial cells. A 3D modeling of the receptor indicates that the receptor has ATP binding sites and TMOF transport into recombinant E. coli cells is inhibited with ATPase inhibitors Na Arsenate and Na Azide.

Mosquitoes are vectors of pathogens affecting humans causing morbidity, mortality and significant economic losses. Insecticides provide main-protection against vector-mosquitoes. However, synthetic insecticides, cause environmental contamination and increase vector resistance. These effects make it a priority to search for alternative means that are not deleterious to the environment and are public health acceptable. Here, we report oviposition deterrence, blood-feeding inhibition, excito-repellent effect, and larvicidal activities by essential oils (EOs) from the leaves of Lantana. camara L., Hyptis. suaveolens Poit., Hyptis. spicigera Lam, and Ocimum. canum Sims against local laboratory strains of Anopheles. gambiae s. s. and Anopheles coluzzii. All bioassays followed WHO standard protocols including minor modifications to evaluate the oviposition deterrence of gravid females, inhibition of adult blood-feeding and mortality caused by EO (essential oils), and larval susceptibility to EO. All EO tested showed strong oviposition deterrence, and the oviposition activity index ranged from −1 to - 0.75. Using a tunnel, we showed that EO have excito-repellent and blood-feeding inhibitory activities, and induced adult mortality. Engorged females tested ranged from 10.58 to 26.40%. L. camara EO had the highest larvicidal activity on both mosquito strains, and LC 50 values of 102.78 and 92.20 ppm (part per million) for An. coluzzii and An. gambiae, respectively. The two mosquito strains showed similar susceptibilities to EOs. Overall, our results show that the EO used in this study could be used in developing effective and environmentally friendly approach to deter oviposition, repel mosquitoes, and control larvae.

Citrus Greening or Huanglongbing (HLB) is a disease of citrus, causing high reduction in citrus production and is transmitted by the Asian citrus psyllid Diaphorina citri Kuwayama vectoring a phloem-limited bacterium Candidatus Liberibacter sp. We report research results using crowdsourcing challenge strategy identifying potential gene targets in D. citri to control the insect using RNA interference (RNAi). From 63 submitted sequences, 43 were selected and tested by feeding them to D. citri using artificial diet assays. After feeding on artificial diet, the three most effective dsRNAs causing 30% mortality above control silenced genes expressing iron-sulfur cluster subunit of the mitochondrial electron transport chain complex (Rieske), heme iron-binding terminal oxidase enzyme (Cytochrome P450) and tetrahydrobiopterin (BH4) pathway enzyme (Pterin 4α-Carbinolamine Dehydratase). These sequences were cloned into a citrus phloem-limited virus (Citrus tristeza virus, CTV T36) expressing dsRNA against these target genes in citrus. The use of a viral mediated “para-transgenic” citrus plant system caused higher mortality to adult D. citri than what was observed using artificial diet, reaching 100% when detached citrus leaves with the engineered CTV expressing dsRNA were fed to adult D. citri. Using this approach, a virus-induced gene silencing (VIGS) can be used to test future transgenic cultivars before genetically engineering citrus. RNA Seq analysis after feeding D. citri CTV-RIE on infected leaves identified transcriptionally modified genes located upstream and downstream of the targeted RIE gene. These genes were annotated showing that many are associated with the primary function of the Rieske gene that was targeted by VIGS.

Trypsin is a serine protease that is synthesized by the gut epithelial cells of female mosquitoes; it is the enzyme that digests the blood meal. To study its molecular regulation, Culex quinquefasciatus late trypsin was purified by diethylaminoethyl (DEAE), affinity, and C18 reverse-phase high performance liquid chromatography (HPLC) steps, and the N-terminal amino acid sequence was determined for molecular cloning. Five overlapping segments of the late trypsin cDNA were amplified by PCR, cloned, and the full sequence (855 bp) was characterized. Three-dimensional models of the pro-trypsin and activated trypsin were built and compared with other trypsin models. Trypsin modulating oostatic factor (TMOF) concentrations in the hemolymph were determined by ELISA and compared with trypsin activity in the gut after the blood meal. The results showed that there was an increase in TMOF concentrations circulating in the hemolymph which has correlated to the reduction of trypsin activity in the mosquito gut. Northern blot analysis of the trypsin transcripts after the blood meal indicated that trypsin activity also followed the increase and decrease of the trypsin transcript. Injections of different amounts of TMOF (0.025 to 50 μg) decreased the amounts of trypsin in the gut. However, Northern blot analysis showed that TMOF injections did not cause a decrease in trypsin transcript abundance, indicating that TMOF probably affected trypsin translation.

Trypsin modulating oostatic factor (TMOF), a decapeptide hormone synthesized by female mosquito ovaries, ganglia and the central nervous system of Aedes aegypti, terminates trypsin biosynthesis in larvae, and blood-fed female mosquitoes. Earlier, TMOF was cloned and expressed as a single copy in Chlorella dessicata and in Saccharomyces cerevisiae cells as a potential larvicide. Here we report the use of a methylotrophic yeast cells, Pichia pastoris, that efficiently express multi copies of heterologous proteins, that are readily ingested by mosquito larvae. P. pastoris was engineered using pPICZB (Invitrogen, CA, United States), and 2 genes: gfp-tmfA and tmfA inserted between KpnI and XbaI in the multiple cloning site. The plasmid carries a strong AOXI promoter and P. pastoris KM71 and KM71H cells were transformed by homologous recombination. The synthesis of GFP-TMOF was followed using UV and clones were analyzed using southern and Northern blot analyses. Cloning tmfA into KM71H and selection on high Zeocin concentration (2.0 mg/mL) identified a clone that carried 10 copies of tmfA. A comparison between a single and high copy (10 genes) insertions using Northern blot analyses showed that a tmfA transcript was highly expressed even after 120 h. SDS-PAGE analysis of KM71 cells transformed with gfp-tmfA identified a protein band that ran at the expected Mr of 31 kDa. Enzyme Linked Immunoadsorbant Assay (ELISA) analysis of the recombinant cells showed that 1.65 × 108 and 8.27 × 107 cells produce 229 and 114 μM of TMOF, respectively, and caused 100% larval mortality when fed to groups of 5 larvae in 25 mL water. These results indicate that the recombinant P. pastoris cells could be used in the future in the marsh to control mosquito populations.