SYSTEMS, METHODS, AND COMPOSITIONS FOR THE CONTROL OF MOSQUITO BLOOD FEEDING BEHAVIOR THROUGH PARATRANSGENIC APPETITE SUPPRESSION

This International PCT Application claims the benefit of and priority to U.S. Provisional Application No. 62/757,816, filed November 9, 2018. The entire specification and figure of the above-referenced application is hereby incorporated, in its entirety by reference.

TECHNICAL FIELD

Generally, the inventive technology relates to novel strategies for the control of mosquito feeding behavior. Specifically, the invention may comprise novel systems, methods, and compositions for control of mosquito blood feeding behavior through paratransgenic appetite suppression.

BACKGROUND

Mosquito-borne diseases are a major public health problem worldwide with no current effective control strategy. One interesting aspect of mosquito biology is the requirement of female mosquitoes to take a blood-meal in order to acquire the necessary nutrients for laying eggs. Thus, blood-feeding is an obligate step in the mosquito lifecycle. Mosquito-borne diseases are transmitted from human to mosquito and from mosquito back to human during blood- feeding; thus making blood-feeding a very important step in the transmission cycle of vector borne diseases. A crucial parameter in the prediction of how efficient a mosquito population is at transmitting pathogens is the rate in which the mosquito bites the human host. Disrupting the human biting rate in mosquitoes that transmit important human pathogen could be a viable strategy for controlling vector borne diseases. Mosquito-borne diseases are transmitted from human to mosquito and from mosquito back to human during blood feeding; thus making blood feeding a very important step in the transmission cycle of vector borne diseases. A crucial parameter in the prediction of how efficient a mosquito population is at transmitting pathogens is the rate in which the mosquito bites the human host, commonly referred to as host seeking. Disrupting the host seeking behavior of mosquitoes that transmit important human pathogen could be a viable strategy for controlling vector borne diseases. After a female mosquito takes a bloodmeal, she does not feed for multiple days until she has completed her gonotrophic cycle.

The evolution of insects to require a blood-meal to complete their lifecycle has evolved multiple times in insects. In the case of mosquitoes, only the female has evolved to require a blood-meal to gain the required nutrients for her eggs to mature. Specifically, after a female mosquito takes a blood-meal, she does not feed for multiple days until she has completed her gonotrophic cycle. It is possible to inject mosquito hemolymph from blood-fed mosquitoes into non blood-fed mosquitoes or to inject a high dose of peptides that activate G protein-coupled neuropeptide Y (NPY)-like receptors and suppress the desire to blood feed. NPY signaling pathways are key regulators of the motivation to feed in many species and highly evolutionarily conserved.

Aedes aegypti has 49 NPY-like receptors, but only one, NPY-like receptor 7 (NPYLR7) has been shown to be involved in the suppression of the desire to take a second blood-meal. NPY signaling pathways are part of G-protein coupled pathways and the downstream transcription factors or target genes are not known. It has also been observed that certain neuropeptides in mosquitoes are expressed as full-length peptides with multiple cleavage sites. These propeptides are then processed into small peptides that interact with receptors in various tissues. The nervous system of Ae. Aegypti is known to penetrate the midgut cells creating a link between the midgut tissue and the nervous system. Peptides can be released from midgut endocrine cells which are activated during blood digestion. These peptides are then excreted into the hemolymph and can act on distant receptors. Expression of the NPYLR7 receptor has been detected in the female abdominal tip and antennae, but this does not exclude its expression in low levels in other tissue.

As such, disruption of a mosquito’s appetite control through paratransgenic supply of NPYLR7 interacting peptides may provide an effective means of vector, and more importantly and vector-borne disease control.

SUMMARY OF THE INVENTION

In one aspect, the present invention includes a novel paratransgenic system which may further include a novel method for implementation of an effector polypeptide-based strategy in which natural mosquito symbiotic bacteria are transformed with plasmids, or stable genome integration, that express effector peptides that may interact with receptors that are part of a mosquito’s appetite control system. In this manner, the effector peptides may help propagate a signal to the target host that suppresses the target’s appetite response. In one aspect, the present invention includes the generation of a novel paratransgenic system for altering the appetite behavior of a mosquito. The invention may specifically include a paratransgenic system configured to deliver one or more effector peptides to a potentially disease-transmitting mosquito that may be seeking a bloodmeal. In one aspect, the invention may include one or more genetically engineered microorganisms configured to deliver one or more effector peptides to a potentially disease-transmitting mosquito that may be configured to disrupt the mosquito’s appetite control and/or perception through propagating an effector peptide activated signal. This signal may cause a mosquito to alter its feeding behavior and cease host-searching for a blood- meal.

In one preferred aspect, the present inventors may generate genetically engineered symbiotic bacteria that are able to colonize the gut and other tissues (endosymbionts) of a target mosquito. As noted above, in one aspect an endosymbiont may be genetically engineered to express and export one or more effector peptides below with affinity for NPYLR7, (SEQ ID NO. 13 (Amino Acid), and SEQ ID NO. 14 (DNA)) to inhibit mosquito feeding, and preferably mosquito feeding in humans. Such peptides may have an affinity for NPYLR7, such that they may bind to, and activate the receptors function in the absence of a blood-meal thereby inhibiting appetite suppression signal pathways in a mosquito. Activation of this signal pathway by the effector protein may cause the host mosquito to alter feeding behavior and cease host-seeking or cease attempts to take a blood-meal, preferably from a human.

In one aspect, the effector peptide coding sequences may be codon optimized for expression in the bacterial host. In this aspect, one or more effector peptides identified below may be operably linked to a promoter and be part of an expression cassette that may be placed into a competent expression vector, such as a plasmid or stably integrated into a bacterial host’s genome. In additional preferred aspect, one or more effector peptides identified below may be modified to include one or more N-terminal secretion signal peptide(s). In one specific aspect, an N-terminal secretion signal peptide may be fused with the effector peptide. In this aspect, N- terminal tagged effector peptides may be secreted to the periplasm of, for example gram-negative bacterial strains for subsequent inclusion in outer-membrane vesicles, or to extra-cellular vesicles of gram positive strains, and released to the extracellular space for uptake by mosquito cells. Cleavage of secretion peptide by bacteria may further ensure delivery of a mature peptide to mosquito cells, mimicking endogenous processing of precursor full length proteins.

In another preferred aspect, an endosymbiont may be genetically engineered to express and export codon optimized A. aegypti effector peptide precursors of neuropeptides. Such effector peptide may be tagged, preferably with an N-terminal secretion peptide identified below to facilitate direct loading to outer membrane vesicles. Uptake of N-terminal tagged effector peptide, also referred to generally as a neuropeptide, by mosquito cells and subsequent processing by endogenous pathways may release the functional processed small effector peptide or neuropeptides. Binding of neuropeptides to neuro-receptor NPYLR7 may activate its signal pathway propagation and suppress female mosquito feeding on human hosts.

The present invention may further include systems, methods and compositions to enhance secretion of one or more effector peptides from a genetically engineered bacterium. In one preferred aspect, a mosquito enteric, symbiotic and/or endosymbiotic may be genetically engineered to express one or more effector peptides. Such genetically engineered bacteria may be further modified to exhibit enhanced or greater than wild-type levels of Outer Membrane Vesicles (OMVs) formation. As a result of this enhanced OMV formation, the secretion of any expressed effector peptides or other target macromolecules from the aforementioned genetically engineered bacteria to the host may be increased.

In one aspect, upregulation of OMV formation may be accomplished through genetically modifying a target bacterium, preferably a symbiotic or endosymbiotic bacteria, to inhibit or eliminate expression of VacJ/Yrb genes. Inhibition or deletion of VacJ/Yrb genes may result in asymmetric accumulation of phospholipids in the outer leaflet of the outer membrane. This asymmetric expansion may further initiate an outward bulging which in turns results in increased secretion.

In another aspect, upregulation of OMV formation may be accomplished through genetically modifying a target bacteria, preferably a symbiotic or endosymbiotic bacteria, to express mutated forms of Lpp and/or the Tol-Pal system of bacteria that may result in inducing the loss of anchoring structures, which in turn increases the rate of OMV secretion.

In still another aspect, upregulation of OMV formation may be accomplished through genetically modifying a target bacteria, preferably a symbiotic or endosymbiotic bacteria, to express mutated forms of one or more enzymes involved in peptidoglycan degradation which may induce OMV formation.

One aspect of the current inventive technology includes strategies to inhibit a target mosquito vector’s desire to take a bloodmeal by manipulating the interaction of one or more neuroreceptors with interacting peptide ligands. One preferred aspect of the current inventive technology includes strategies to inhibit a target mosquito vector’s desire to take a bloodmeal by manipulating the interaction of the NPYLR7 receptor (SEQ ID NO. 13) with interacting peptide ligands. This may be accomplished by genetically engineering symbiotic, endosymbiotic, and/or enteric bacteria, such as select strains of Enterobacter or E. coli that may be present outside of the gut of Ae. Aegypti to express selected effector peptides that interact with NPYLR7 in the absence of a blood meal. This interaction may propagate a signal pathway to the target host that suppresses the target’s appetite response. A reduction in a target mosquito blood feeding behavior may form a vector borne disease biocontrol strategy by reducing the number of human- vector interactions. By reducing such interaction, the current system may reduce the transmission of vector borne diseases, such as dengue, Zika, malaria, yellow fever and the like.

Another aspect of the invention includes systems and methods for the paratransgenic supply of NPYLR7 interacting peptides. In one preferred aspect, full length NPYLR7 interacting peptides may be expressed and delivered by a symbiotic or probiotic bacteria, for example, and further processed into small NPYLR7 interacting or effector peptides that may propagate a NPYLR7-directed signal pathway to the target host that suppresses the target’s appetite response, and in turn reduce the number of human bloodmeal interactions.

Another aspect may include one or more symbiotic bacteria that may be genetically engineered to express one or more NPYLR7 effector peptides. In this preferred aspect, one or more full-length NPYLR7 effector peptides may be part of an expression cassette that is operably linked to a promotor. In another aspect, one or more full-length NPYLR7 effector peptides may be coupled with a secretion signal, such as a TAT secretion signal, to have improved secretion out of the bacteria and into the target host where they may interact with the NPYLR7 receptor and propagate a signal pathway to the target host that suppresses the target’ s appetite response.

Another aspect may include a treated feed or other composition that may be introduced to the environment, or to a mosquito population that may contain one or more symbiotic bacteria that may be genetically engineered to express one or more NPYLR7 effector peptides as generally described herein.

Additional aspects of the invention may include one or more of the following preferred embodiments:

1. A paratransgenic method of controlling blood feeding behavior in a mosquito comprising the steps of: introducing into the mosquito a genetically modified bacteria that colonizes the mosquito, wherein the bacteria comprises an expression control sequence operably linked to a nucleotide sequence encoding one or more heterologous effector polypeptides that may be delivered to the mosquito and processed into one or more small effector peptide ligands; and

wherein said one or more small effector peptide ligands interact with at least one neuroreceptor in the mosquito propagating a signal pathway that suppresses the mosquito’s appetite response resulting in fewer bloodmeals.

2. The method of embodiment 1, wherein said at least one neuroreceptor comprises NPYLR7.

3. The method of embodiments 1 or 2, wherein said at least one neuroreceptor comprises neuroreceptor according to amino acid sequence SEQ ID NO. 13.

4. The method of embodiments 2 or 3, wherein the mosquito is a female mosquito that transmits the virus to a mammalian organism.

5. The method of embodiment 4, wherein the introducing to a mosquito the genetically modified bacteria comprises a method selected from the group consisting of: soaking, spraying, injecting, feeding, introducing to through an aerosolized disbursement, introducing to through an environmental aerosolized disbursement, introducing to through an environmental aerosolized disbursement in water sources, brushing, dressing, dripping, and coating the mosquito and wherein during the introduction the bacteria is lyophilized, freeze-dried, microencapsulated, desiccated, in an aqueous carrier or in a solution.

6. The method of embodiments 2, wherein the genetically modified bacteria is obtained from bacteria selected from the group consisting of: an endosymbiotic bacteria that persists through the life-cycle of the mosquito, Enterobacter strain Ae073, and E. coli strain HT27.

7. The method of embodiments 2 or 3, wherein said one or more heterologous effector polypeptides comprises one or more heterologous effector polypeptides selected from the group consisting of: FMRFa, MIP-l, Leucokinin, and AAEL011702A, or a combination thereof.

8. The method of embodiments 2 or 3, wherein said one or more heterologous effector polypeptides is selected from the amino acid sequences of SEQ ID NOs. 2, 4, 6, and 8, or a combination thereof. 9. The method of embodiments 2 or 3, wherein said one or more small effector peptide ligands is selected from the group consisting of: FMRFa-3 processed peptide, FMRFa-l processed peptide, FMRFa-lO processed peptide, MIP-l processed peptide, Leucokinin-3 processed peptide, and AAEL011702A processed peptide, or a combination thereof.

10. The method of embodiments 2 or 3, wherein said one or more small effector peptide ligands is selected from the amino acid sequences of SEQ ID NOs. 15-20.

11. The method of embodiments 2 or 3, wherein said one or more heterologous effector polypeptides is fused with a secretion peptide.

12. The method of embodiment 11, wherein said secretion peptide comprises a TAT secretion peptide.

13. The method of embodiment 12, wherein said a TAT secretion peptide comprises a TorA secretion peptide according to amino acid sequence SEQ ID NO. 12, or an OmpA sequence secretion peptide according to amino acid sequence SEQ ID NO. 9.

14. The method of embodiments 2 or 3, wherein said one or more small effector peptide ligands is fused with a secretion peptide.

15. The method of embodiment 14, wherein said secretion peptide comprises a TAT secretion peptide.

16. The method of embodiment 15, wherein said a TAT secretion peptide comprises a TorA secretion peptide according to amino acid sequence SEQ ID NO. 12, or an OmpA sequence secretion peptide according to amino acid sequence SEQ ID NO. 9.

17. The method of embodiments 2 or 3, wherein said nucleotide sequence encoding one or more heterologous effector polypeptides is selected from one of the nucleotide sequences of SEQ ID NOs. 1, 3, 5, and 7, or a combination thereof.

18. The method of embodiments 2 or 3, wherein the suppression of the mosquito’s appetite response resulting in fewer bloodmeals comprises a reduction in the number of human bloodmeals taken by the mosquito.

19. The method of embodiments 2 or 3, wherein said one or more heterologous effector polypeptides that may be delivered to the mosquito and processed into one or more small effector peptide ligands comprises one or more heterologous effector polypeptides that may be delivered to the mosquito through outer membrane vesicles (OMVs) and processed into one or more small effector peptide ligands. 20. A genetically modified bacteria that is paratransgenic with a mosquito, comprising an expression control sequence operably linked to a nucleotide sequence encoding one or more heterologous effector polypeptides that may be delivered to the mosquito and processed into one or more small effector peptide ligands, and wherein said one or more small effector peptide ligands interact with at least one neuroreceptor in the mosquito propagating a signal pathway that suppresses the mosquito’s appetite response resulting in fewer bloodmeals.

21. The genetically modified bacteria of embodiment 20, wherein said at least one neuroreceptor comprises NPYLR7.

22. The genetically modified bacteria of embodiments 20 or 21, wherein said at least one neuroreceptor comprises neuroreceptor according to amino acid sequence SEQ ID NO. 13.

23. The genetically modified bacteria of embodiments 21 or 22, wherein the mosquito is a female mosquito that transmits the virus to a mammalian organism.

24. The genetically modified bacteria of embodiment 23, wherein the introducing to a mosquito the genetically modified bacteria comprises a method selected from the group consisting of: soaking, spraying, injecting, feeding, introducing to through an aerosolized disbursement, introducing to through an environmental aerosolized disbursement, introducing to through an environmental aerosolized disbursement in water sources, brushing, dressing, dripping, and coating the mosquito and wherein during the introduction the bacteria is lyophilized, freeze-dried, microencapsulated, desiccated, in an aqueous carrier or in a solution.

25. The genetically modified bacteria of embodiment 21, wherein the genetically modified bacteria is obtained from bacteria selected from the group consisting of: an endosymbiotic bacteria that persists through the life-cycle of the mosquito, Enterobacter strain Ae073, and A. coli strain HT27.

26. The genetically modified bacteria of embodiments 21 or 22, wherein said one or more heterologous effector polypeptides comprises one or more heterologous effector polypeptides selected from the group consisting of: FMRFa, MIP-l, Leucokinin, and AAEL011702A, or a combination thereof.

27. The genetically modified bacteria of embodiments 21 or 22, wherein said one or more heterologous effector polypeptides is selected from the amino acid sequences of SEQ ID NOs. 2, 4, 6, and 8, or a combination thereof. 28. The genetically modified bacteria of embodiments 21 or 22, wherein said one or more small effector peptide ligands is selected from the group consisting of: FMRFa-3 processed peptide, FMRFa-l processed peptide, FMRFa-lO processed peptide, MIP-l processed peptide, Leucokinin-3 processed peptide, and AAEL011702A processed peptide, or a combination thereof.

29. The genetically modified bacteria of embodiments 21 or 22, wherein said one or more small effector peptide ligands is selected from the amino acid sequences of SEQ ID NOs. 15-20.

30. The genetically modified bacteria of embodiments 21 or 22, wherein said one or more heterologous effector polypeptides is fused with a secretion peptide.

31. The genetically modified bacteria of embodiment 30, wherein said secretion peptide comprises a TAT secretion peptide.

32. The genetically modified bacteria of embodiment 31, wherein said a TAT secretion peptide comprises a TorA secretion peptide according to amino acid sequence SEQ ID NO. 12, or an OmpA sequence secretion peptide according to amino acid sequence SEQ ID NO. 9.

33. The genetically modified bacteria of embodiments 21 or 22, wherein said one or more small effector peptide ligands is fused with a secretion peptide.

34. The genetically modified bacteria of embodiment 33, wherein said secretion peptide comprises a TAT secretion peptide.

35. The genetically modified bacteria of embodiment 34 wherein said a TAT secretion peptide comprises a TorA secretion peptide according to amino acid sequence SEQ ID NO. 12, or an OmpA sequence secretion peptide according to amino acid sequence SEQ ID NO. 9.

36. The genetically modified bacteria of embodiments 21 or 22, wherein said nucleotide sequence encoding one or more heterologous effector polypeptides is selected from one of the nucleotide sequences of SEQ ID NOs. 1, 3, 5, and 7, or a combination thereof.

37. The genetically modified bacteria of embodiments 21 or 22, wherein the suppression of the mosquito’s appetite response resulting in fewer bloodmeals comprises a reduction in the number of human bloodmeals taken by the mosquito.

38. The genetically modified bacteria of embodiments 21 or 22, wherein said one or more heterologous effector polypeptides that may be delivered to the mosquito and processed into one or more small effector peptide ligands comprises one or more heterologous effector polypeptides that may be delivered to the mosquito through outer membrane vesicles (OMVs) and processed into one or more small effector peptide ligands.

39. A method of controlling vector-born disease comprising the steps of:

introducing into the mosquito population a genetically modified bacteria that colonizes the mosquito population, wherein the bacteria comprises an expression control sequence operably linked to a nucleotide sequence encoding one or more heterologous effector polypeptides that may be delivered to individual mosquitos in the population and processed into one or more small effector peptide ligands; and

- wherein said one or more small effector peptide ligands interact with at least one neuroreceptor in the individual mosquitos in the population propagating a signal pathway that suppresses the mosquito’s appetite response resulting in the taking of fewer bloodmeals in humans thereby reducing the transmission of vector borne-disease by the mosquito population.

40. The method of embodiment 39, wherein said at least one neuroreceptor comprises NPYLR7.

41. The method of embodiments 39 or 40, wherein said at least one neuroreceptor comprises neuroreceptor according to amino acid sequence SEQ ID NO. 13.

42. The method of embodiments 40 or 41, wherein the mosquito is a female mosquito that transmits the virus to a mammalian organism.

43. The method of embodiment 42, wherein the introducing to a mosquito the genetically modified bacteria comprises a method selected from the group consisting of: soaking, spraying, injecting, feeding, introducing to through an aerosolized disbursement, introducing to through an environmental aerosolized disbursement, introducing to through an environmental aerosolized disbursement in water sources, brushing, dressing, dripping, and coating the mosquito and wherein during the introduction the bacteria is lyophilized, freeze-dried, microencapsulated, desiccated, in an aqueous carrier or in a solution.

44. The method of embodiments 40, wherein the genetically modified bacteria is obtained from a bacteria selected from the group consisting of: an endosymbiotic bacteria that persists through the life-cycle of the mosquito, Enterobacter strain Ae073, and E. coli strain HT27.

45. The method of embodiments 40 or 41, wherein said one or more heterologous effector polypeptides comprises one or more heterologous effector polypeptides selected from the group consisting of: FMRFa, MIP-l, Leucokinin, and AAEL011702A, or a combination thereof.

46. The method of embodiments 40 or 41, wherein said one or more heterologous effector polypeptides is selected from the amino acid sequences of SEQ ID NOs. 2, 4, 6, and 8, or a combination thereof.

47. The method of embodiments 40 or 41, wherein said one or more small effector peptide ligands is selected from the group consisting of: FMRFa-3 processed peptide, FMRFa- 1 processed peptide, FMRFa- 10 processed peptide, MIP-l processed peptide, Leucokinin-3 processed peptide, and AAEL011702A processed peptide, or a combination thereof.

48. The method of embodiments 40 or 41, wherein said one or more small effector peptide ligands is selected from the amino acid sequences of SEQ ID NOs. 15-20.

49. The method of embodiments 40 or 41, wherein said one or more heterologous effector polypeptides is fused with a secretion peptide.

50. The method of embodiment 49, wherein said secretion peptide comprises a TAT secretion peptide.

51. The method of embodiment 50, wherein said a TAT secretion peptide comprises a TorA secretion peptide according to amino acid sequence SEQ ID NO. 12, or an OmpA sequence secretion peptide according to amino acid sequence SEQ ID NO. 9.

52. The method of embodiments 40 or 41, wherein said one or more small effector peptide ligands is fused with a secretion peptide.

53. The method of embodiment 52, wherein said secretion peptide comprises a TAT secretion peptide.

54. The method of embodiment 53 wherein said a TAT secretion peptide comprises a TorA secretion peptide according to amino acid sequence SEQ ID NO. 12, or an OmpA sequence secretion peptide according to amino acid sequence SEQ ID NO. 9.

55. The method of embodiments 40 or 41, wherein said nucleotide sequence encoding one or more heterologous effector polypeptides is selected from one of the nucleotide sequences of SEQ ID NOs. 1, 3, 5, and 7, or a combination thereof.

56. The method of embodiments 40 or 41, wherein the suppression of the mosquito’s appetite response resulting in fewer bloodmeals comprises a reduction in the number of human bloodmeals taken by the mosquito. 57. The method of embodiments 40 or 41, wherein said one or more heterologous effector polypeptides that may be delivered to the mosquito and processed into one or more small effector peptide ligands comprises one or more heterologous effector polypeptides that may be delivered to the mosquito through outer membrane vesicles (OMVs) and processed into one or more small effector peptide ligands.

58. The method of embodiments 40 or 41, wherein the reduction in vector borne-disease transmission comprises a reduction one or more of the vector borne-diseases selected from the group consisting of: a Zika virus, La Crosse encephalitis virus, an Eastern equine encephalitis virus, a Japanese encephalitis virus, a Western equine encephalitis virus, a St. Louis encephalitis virus, a Tick-borne encephalitis virus, a Ross River virus, a Venezuelan equine encephalitis virus, a Chikungunya virus, a West Nile virus, a Dengue virus, a Yellow fever virus, a Bluetongue disease virus, a Sindbis Virus, a Rift Valley Fever virus, a Colorado tick fever virus, a Murray Valley encephalitis virus, an Oropouche virus, and a Flock House virus.

Other features and advantages of the invention will be apparent from the detailed description, and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

The novel aspects, features, and advantages of the present disclosure will be better understood from the following detailed descriptions taken in conjunction with the accompanying figures, all of which are given by way of illustration only, and are not limiting the presently disclosed embodiments, in which:

FIG. 1: Female mosquitoes 5-7 days old were injected with 1 ng of AGQGFMRF, PFFVGSRY, TWKNLQGGW signaling peptide or PBS. The proportion of mosquitoes taking a bloodmeal 24 hours post injection is shown and represents 2 biological replicate experiments. All 3 signaling peptides are significantly different than the PBS injected mosquitoes by Chisq (TWK: p-value = 0.03, AGQ: p-value = 0.019, PFF: p-value = 0.009)

FIG. 2: Plasmid map for PFFVGRSY constructs with the OX19 promoter; and OmpA secretion peptide in the (A) OX, and (B) Tn7 backbone

FIG. 3: Gel picture of stable integration of PFFVGSRY + OmpA secretion peptide in Enterobacter. Colony PCR analysis of TN7 integrated Enterobacter Ae073 with small peptide PFFVGSRY. Expected size is 247 base pairs. Clones 3 and 5 were chosen for glycerol stock and mosquito feeding. FIG. 4: Mass spectrophotometry showing PFFVGSRY export from Enterobacter. See also Table 6.

FIG. 5: (Al) Plasmid maps for AAEL011702A in OX1 backbone; (A2) Plasmid maps for TN7 backbone; (Bl) Plasmid maps for FMRFa in OX1 backbone; (B2) Plasmid maps for TN7 backbone; (Cl) Plasmid maps for Leucokinin in OX1 backbone; and (C2) Plasmid maps for TN7 backbone; (Dl) Plasmid maps for MIP in OX1 backbone; (D2) Plasmid maps for TN7 backbone

FIG. 6: Gel picture of the stable integration of AAEL011702A into Enterobacter. Colony PCR analysis of TN7 integrated Enterobacter Ae073 with AAEL011702A. Expected size is 247 base pairs. Clones 2, 4, 6, and 8 were chosen for glycerol stock and mosquito feeding.

FIG. 7: Gel picture of the stable integration of FMRFa into Enterobacter. Colony PCR analysis of TN7 integrated Enterobacter Ae073 with FMRFa. Expected size is 247 base pairs. Colonies 3, 4, 6, 7, and 8 were chosen for glycerol stock and clone 8 was used for mosquito feeding.

FIG. 8: Gel picture of the stable integration of Leucokinin into Enterobacter. Colony PCR analysis of TN7 integrated Enterobacter Ae073 with Leucokinin. Expected size is 247 base pairs. Clone 3 was chosen for glycerol stock and mosquito feeding.

FIG. 9: Gel picture of the stable integration of MIP into Enterobacter. Colony PCR analysis of TN7 integrated Enterobacter Ae073 with MIP. Expected size is 247 base pairs. Clone 7 was chosen for glycerol stock and mosquito feeding.

FIG. 10: Confirmation of gene expression of Leucokinin in Enterobacter Ae073 cells. Western blot showed expression of two clones of Leucokinin protein size 27.3 kDa in Enterobacter Ae073 cells. The blot was probed with anti Flag antibody. The two clones of Leucokinin showed good protein expression. A construct with a Flag-tag used as positive and a construct lacking a FLAG- tag as negative control.

FIG. 11 A: Quantification of GFP transcripts in the mosquito following bacteria feed delivery loquacious protein and dsGFP. Adult mosquitoes which express GFP in the midgut following a bloodmeal fed a solution without bacteria or with E. colt cells delivering dsRNA- GFP or E. colt cells delivering dsRNA-GFP and the Loqs protein. The mosquitoes either received a bloodmeal (BF) or not (NBF) 48 hours post bacteria feed and the mosquitoes were harvested 24 hours later. RNA was extracted from pools of 5 mosquitoes and the copies of GFP cDNA (Copies GFP cDNA/ul) was quantified by digital droplet PCR. Each sample above represents a pool of 5 mosquitoes. Based on these results we conclude that the co-expression of the Loqs protein enhances the silencing of GFP by dsRNA-GFP compared to delivery of just the dsRNA-GFP alone

FIG. 12A-B: (A) Reduction in blood feeding behavior after bacterial delivery of a cocktail of all 4 full-length proteins. Aedes aegypti larval were hatched in sterile water and reared in water inoculated (OD 0.7) with an Enterobacter strain containing an empty vector or delivering the appetite control proteins. The desire to take a bloodmeal was measure in 30-40 adult females 5-7 days post emergence. Based on these results we see that inoculating larvae with a symbiotic bacteria delivering an appetite control protein makes them less likely to take a bloodmeal compared to mosquito inoculated with an empty vector. Data represents three independent biological replicates. (B) Mass Spec detection of processed peptide after feeding the full-length proteins.

FIG. 13: Detecting processed peptides in the mosquito by Mass spec after delivery of full-length protein. Relative amount (ng) of signaling peptide observed in protein extracts from bacteria-fed mosquitoes. Relative amounts were calculated from synthetic standards, and 100 fmol/pL Glul-fibrinopeptide B was used as an internal standard to calculate calibration response factor (CRF).

FIG. 14A-D: Relative amounts of processed peptides in the mosquito by Mass spec after delivery of full-length protein. Relative amount (ng) of signaling peptide observed in protein extracts from bacteria-fed mosquitoes fed (A-B) FMRFa, (C) Leucokinin, or (D) AAEL011702A. Relative amounts were calculated from synthetic standards, and 100 fmol/pL Glul-fibrinopeptide B was used as an internal standard to calculate calibration response factor (CRF). The first three letters of the amino acid sequence are used as an abbreviation.

FIG. 15A-B: Plasmid maps of (A) AAEL011702A, and (B) FMRFa constructs in the OX11 backbone and the TorA TAT secretion peptide

FIG. 16: Gene expression confirmation of AAEL011702A and FMRFA in HT27 cells in constructs with the TorA TAT secretion peptide. Western blot shows protein expression of AAEL011702A and FMRFA in HT27 cells. The blot was probed with anti-HA antibody. Expected size of FmRFa is 47.4 Kda and Aael is 25.2 KDa FIG. 17: Gene expression confirmation of MIP and FMRFA in HT27 without a TAT secretion peptide. Western blot shows expression of two peptides FMRFa and MIP in HT27 cells. The blot was probed with anti-Flag antibody. A Flag-tagged protein was used as positive control and PFFVGSRY construct in the OX1 backbone with no Flag-tag was used as a negative control. Expected size of FmRFa is 40.4KDa and MIP is 24.4 KDa.

FIG. 18: Gene expression confirmation of MIP and Leucokinin in HT27 without a TAT secretion peptide.

FIG. 19: Gene expression confirmation of FMRFa delivered from HT27 cells in the mosquito. Western blot showing expression of FMRFa in the mosquito following bacterial feeding at the adult stage of HT27 cells delivering FMRFa or AAEL011702A under the OX11 promoter with the TorA secretion peptide. The part of the membrane containing the positive control was cut off to do a long exposure to detect the protein.

FIG. 20A-B: (A) Reduction in blood feeding behavior after bacterial delivery of full- length MIP protein. Adult Ae. aegypti from the MLM colony of mosquitoes were starved for 24 hours and then offered a feeding solution either containing no bacteria or HT27 cells delivering the MIP protein under the OX1 promotor. Mosquitoes were allowed to feed on the bacteria for 24 hours and then given access to sugar solution. The desire to take a bloodmeal was test seven days after bacteria ingestion. (B) Statistical analysis for data presented in FIG. 20.

FIG. 21A-B: Mass Spec data showing processed MIP in mosquito. Relative amount (ng) of signaling peptide observed in protein extracts from mosquitoes fed MIP at the adult stage. Relative amounts were calculated from synthetic standards, and 100 fmol/pL Glul- fibrinopeptide B was used as an internal standard to calculate calibration response factor (CRF); Mass Spec data showing processed MIP in mosquito. Relative amount (ng) of signaling peptide observed in protein extracts from mosquitoes fed MIP at the adult stage. Relative amount (ng) of signaling peptide observed in protein extracts from mosquitoes fed MIP. Relative amounts were calculated from synthetic standards, and 100 fmol/pL Glul-fibrinopeptide B was used as an internal standard to calculate calibration response factor (CRF).

FIG. 22A-B: (A) Enhanced reduction in blood feeding behavior after inclusion of a TorA secretion peptide on the full-length FMRFa protein . Adult Ae. aegypti from the MLM colony of mosquitoes were starved for 24 hours and then offered a feeding solution either containing no bacteria, HT27 cells delivering the FMRFa protein under the OX1 promotor, or HT27 cells delivering the FMRFa protein with a TorA secretion peptide under the 0X11 promoter. Mosquitoes were allowed to feed on the bacteria for 24 hours and then given access to sugar solution. The desire to take a bloodmeal was test seven days after bacteria ingestion. (B) Statistical analysis for data presented in FIG. 22. See also Table 7a-b.

FIG. 23: Relative abundance of AAEL011702A protein in mosquitoes following bacterial ingestion by Mass Spectrophotometry. Normalized abundance of AAEL011702A detected in bacteria-fed larvae. Data are normalized to peptide signal intensity for peptides with non-conflicting protein identifications.

FIG. 24: Coverage map of peptides aligning to AAEL011702A by Mass Spectrophotometry. Protein sequence and % coverage of AAEL011702A obtained from analysis of protein digest by LC-MS/MS. Three peptides were identified for 16.45% overall coverage for SEQ ID NO 2.

FIG. 25: Relative abundance of MPM protein in mosquitoes following bacterial ingestion by Mass Spectrophotometry. Normalized abundance of MPM detected in bacteria-fed adult mosquitoes. Data are normalized to peptide signal intensity for peptides with non- conflicting protein identifications.

FIG. 26: Coverage map of peptides aligning to MIP-l by Mass Spectrophotometry. Protein sequence and % coverage of MIP-l obtained from analysis of protein digest by LC- MS/MS. Three peptides were identified for 15.92% overall coverage of SEQ ID NO. 8.

FIG. 27: Detecting processed peptides in the mosquito by Mass spec after delivery of all four full-length proteins.

FIG. 28A-B: Detecting processed peptides in the mosquito by Mass spec after delivery of full-length protein. (A) Mass Spec detection of processed peptide after feeding the full-length protein. (B) Comparison of the abundance of the small peptides after ingestion of the FMRFa protein with and without TorA secretion peptide.

DETAILED DESCRIPTION OF INVENTION

In one embodiment the present invention includes a novel paratransgenic system which may further include a novel method for implementation of an effector peptide-based strategy in which natural mosquito symbiotic bacteria are transformed with plasmids, or stable genome integration that express effector peptides that may interact with receptors that are part of a mosquito’s appetite control system. In this manner, the effector peptides may help propagate a signal to the target host that suppresses the target’s appetite response. In one embodiment the present invention includes the generation of a novel paratransgenic system for altering the appetite behavior of a mosquito. The invention may specifically include a paratransgenic system configured to deliver one or more effector peptides to a potentially disease-transmitting mosquito that may be seeking a bloodmeal.

In one embodiment, the invention may include one or more genetically engineered microorganisms configured to deliver one or more effector peptides to a potentially disease- transmitting mosquito that may be configured to disrupt the mosquito’s appetite control and/or perception through propagating an effector peptide activated signal through a NPYLR7 receptor. This signal may cause a mosquito to alter its feeding behavior and cease host-searching for a blood-meal. In certain embodiments, such effector peptides may be fused with secretion signal, preferably through a fusion peptide that may facilitate enhanced secretion from the bacterial source and into the target mosquito host. In this embodiment, the colonized bacteria may express effector peptides that may bind to receptors that are part of a mosquito’s appetite control. This interaction may sterically or competitively activate the receptor’s normal function, which may propagate a corresponding appetite control signal pathway the NPYLR7 receptor. Moreover, these colonized symbiotic bacteria, having become a part of the host’s natural microbiome, may continuously deliver, for example, effector peptide molecules via the intestine from the earliest larval stages to the adult stage providing appetite control-specific signal propagation throughout the host’s lifecycle. In addition, as the symbiotic bacteria vector may be an already naturally occurring part of the host’s microbiome, its presence may not pose any risk to the organism, environment or end-consumers.

The present invention may further include one or more vectors for modulating appetite control in a target host, wherein the vector may encode one, or a plurality of effectors peptides that may inhibit appetite in a host. Such effectors peptides may be pre-processed as discussed below. In one embodiment, such effectors peptides may include one or more of the following full-length proteins: AAEL011702A (SEQ ID NO. 2), FMRFa (SEQ ID NO. 4), Leucokinin (SEQ ID NO. 6), and MIP (SEQ ID NO. 8). This embodiment may include the use of a plasmid expression system. In some embodiments, this plasmid may have one or more expression cassettes, including: at least one gene suppressing cassette containing a polynucleotide operably- linked to a promoter sequence, wherein the polynucleotide encodes an effector peptide molecule, for example one or more of the following nucleotide sequences: AAEL011702A (SEQ ID NO. 1), FMRFa (SEQ ID NO. 3), Leucokinin (SEQ ID NO. 5), and MIP (SEQ ID NO. 7), that target appetite control receptors and cause a signal propagation that alters a target mosquito’s blood feeding and/or feeding behavior.

The present invention may further include one or more vectors for modulating appetite control in a target host, wherein the vector may encode one, or a plurality of full-length effectors peptides that may be processed into small effector peptides that may interact with the NPYLR7 receptor and may inhibit appetite in a host. In one embodiment, such processed small peptide effectors may include one or more of the following small peptides: FMRFa-3 process peptide sequence (SEQ ID NO. 15), FMRFa-l process peptide sequence (SEQ ID NO. 16), FMRFa-lO process peptide sequence (SEQ ID NO. 17), MIP-l process peptide sequence (SEQ ID NO. 18), Leucokinin-3 process peptide sequence (SEQ ID NO. 19), and AAEL011702A (SEQ ID NO. 20).

This embodiment may include the use of a plasmid expression system. In some embodiments, this plasmid may have one or more expression cassettes, including: at least one gene suppressing cassette containing a polynucleotide operably-linked to a promoter sequence, wherein the polynucleotide encodes an effector peptide molecule, for example one or more of the following nucleotide sequences: AAEL011702A (SEQ ID NO. 1), FMRFa (SEQ ID NO. 3), Leucokinin (SEQ ID NO. 5), and MIP (SEQ ID NO. 7). Such full-length effector proteins may be expressed in symbiotic bacteria, such as Enterobacter strain Ae073, or E. coli strain HT27, or one or more strains identified in Tables 1A-1B of US Provisional Application No. 62/757,816, incorporated herein by reference, and transported to a target host, in this case a mosquito through one or more OMVs. Once delivered to the host, the full-length effector proteins may be processed and cleaved into small effector peptides (e.g. SEQ ID NOs. 15-20) that may specifically interact with the NPYLR7 receptor and cause a signal propagation that reduce a target mosquito’s blood feeding and/or feeding behavior below wild-type levels.

The present invention may further include one or more vectors for modulating appetite control in a target host, wherein the vector may encode one, or a plurality of effectors peptides that may inhibit appetite in a host, wherein one or more of the effectors peptide may be fused with a secretion signal to enhance export from a bacterial expression vector to a target host, in this case a mosquito host. In one embodiment, such effectors peptides may include one or more of the following full-length proteins: AAEL011702A (SEQ ID NO. 2), FMRFa (SEQ ID NO. 4), Leucokinin (SEQ ID NO. 6), and MIP (SEQ ID NO. 8), which may further be fused with a TAT secretion signal, and preferably a TorA secretion peptide (SEQ ID NO. 11), and a OmpA sequence secretion peptide (SEQ ID NO. 9). In another embodiment, such effectors peptides may include one or more of the following small peptides: FMRFa-3 process peptide sequence (SEQ ID NO. 15), FMRFa-l process peptide sequence (SEQ ID NO. 16), FMRFa-lO process peptide sequence (SEQ ID NO. 17), MIP-l process peptide sequence (SEQ ID NO. 18), Leucokinin- 3 process peptide sequence (SEQ ID NO. 19), and AAEL011702A (SEQ ID NO. 20), which may further be fused with a TAT secretion signal, and preferably a TorA secretion peptide (SEQ ID NO. 11), or a OmpA sequence secretion peptide (SEQ ID NO. 9). Expression of such fusion proteins may be under the control of a promoter, and may further be codon-optimized for expression in one or more endosymbiotic bacteria. Additional examples of N-terminal peptides to be fused to peptides / proteins engineered into enteric bacteria for secretion may include one or more of the signal peptides identified in Table 3 of US Provisional Application No. 62/757,816, incorporated herein by reference.

In yet another embodiment, the current invention may include systems, methods and compositions for increasing the efficiency of select effector peptide or other macromolecule secretion from a genetically modified bacterium. In this preferred embodiment, a genetically modified target bacteria, preferably a symbiotic or endosymbiotic bacteria, may be configured to lack certain genes, such as VacJ/Yrb genes, that may upregulate Outer Membrane Vesicles (OMVs) formation and thus facilitation excretion of any expressed effector peptides or other target macromolecules from the aforementioned genetically engineered bacteria to the host. In another embodiment, a genetically modified target bacteria, preferably a symbiotic or endosymbiotic bacteria, may be configured to express mutant exogenous genes, such as Lpp and/or the Tol-Pal system of bacteria, or one or more enzymes involved in peptidoglycan degradation. Expression of such mutant exogenous genes facilitation excretion of any expressed effector peptides or other target macromolecules from the aforementioned genetically engineered bacteria to the host.

The term“mosquito” or“mosquitoes” as used herein, refers to an insect of the family Culicidae. The mosquito of the invention may include an adult mosquito, a mosquito larva, a pupa or an egg thereof. Typically, a mosquito’s life cycle includes four separate and distinct stages: egg, larva, pupa, and adult. Thus, a mosquito’s life cycle begins when eggs are laid on a water surface (e.g. Culex, Culiseta, and Anopheles species) or on damp soil that is flooded by water (e.g. Aedes species). Most eggs hatch into larvae within 48 hours. The larvae live in the water, feeding on microorganisms and organic matter and come to the surface to breathe. They shed their skin four times, growing larger after each molting, and on the fourth molt the larva changes into a pupa. The pupal stage is a resting, non-feeding stage of about two days. At this time the mosquito turns into an adult. When development is complete, the pupal skin splits and the mosquito emerges as an adult.

According to one embodiment, the mosquitoes are of the sub-families Anophelinae and Culicinae. According to one embodiment, the mosquitoes are of the genus Culex, Culiseta, Anopheles and Aedes. Exemplary mosquitoes include, but are not limited to, Aedes species e.g. Aedes aegypti, Aedes albopictus, Aedes polynesiensis, Aedes australis, Aedes cantator, Aedes cinereus, Aedes rusticus, Aedes vexans', Anopheles species e.g. Anopheles gambiae, Anopheles freeborni, Anopheles arabiensis, Anopheles funestus, Anopheles gambiae Anopheles moucheti, Anopheles balabacensis, Anopheles baimaii, Anopheles culicifacies, Anopheles dims, Anopheles latens, Anopheles leucosphyrus, Anopheles maculatus, Anopheles minimus, Anopheles fluviatilis sd., Anopheles sundaicus Anopheles superpictus, Anopheles farauti, Anopheles punctulatus, Anopheles sergentii, Anopheles stephensi, Anopheles sinensis, Anopheles atroparvus, Anopheles pseudopunctipennis, Anopheles bellator and Anopheles cruzii', Culex species e.g. C. annulirostris, C. antennatus, C. jenseni, C. pipiens, C. pusillus, C. quinquefasciatus, C. rajah, C. restuans, C. salinarius, C. tarsalis, C. territans, C. theileri and C. tritaeniorhynchus; and Culiseta species e.g. Culiseta incidens, Culiseta impatiens, Culiseta inornata and Culiseta particeps.

Paratransgenesis is generally understood as a technique that attempts to eliminate or reduce a pathogen from vector populations through transgenesis of a symbiont of the vector. The goal of this technique is to control vector-borne diseases, for example through the reduction in the number of bloodmeals a mosquito might take, thereby reducing the overall number of vector- borne disease transmission events that may occur within a population. The first step is to identify proteins that prevent the vector species from transmitting the pathogen. The genes coding for these proteins are then introduced into the symbiont, so that they can be expressed in the vector. The final step in the strategy is to introduce these transgenic symbionts into vector populations in the wild. Characteristics of a successful paratransgenesis system may include one or more of the following: The symbiotic bacteria can be grown in vitro easily; They can be genetically modified, such as through transformation with a plasmid containing the desired gene; The engineered symbiont is stable and safe; The association between vector and symbiont cannot be attenuated; Field delivery is easily handled; A paratransgenic system is a system that can achieve paratransgenesis in a target organism.

Identification of suitable commensal bacteria that are non-pathogenic to humans or animals among the many organisms that insects harbor, particularly in their digestive systems, is paramount for the success of a paratransgenic system. In mosquitoes, these bacteria are involved in various biological functions associated with digestion, primarily in the midgut. There is a close association between blood-dependent insects and symbiotic microorganisms that help the anabolic processes of vitellogenesis and ovogenesis. Eradication of these bacteria leads to a decline in fecundity and a slower growth rate. Interference with the digestion of proteins in mosquito blood meals can reduce fecundity and may represent a new approach for controlling mosquito populations and preventing the transmission of pathogens.

For example, the chosen bacteria should be capable of colonizing a wide variety of mosquito species so that they can be deployed in different species and isolated strains. Furthermore, the number of bacteria increases dramatically (100 to 1000 of times) after ingestion of blood, resulting in a proportional increase in the amount of effector molecules expressed and secreted by GM bacteria, leading to various possible outcomes: obstructing pathogen transmission, reducing the mosquito's vector capacity, preventing fertilization of eggs, interfering with embryogenesis and causing the death of the mosquito. These technical and physiological challenges make the development of paratransgenic systems extremely difficult. Importantly, these technical issues are such that many paratransgenic systems are neither effective nor appropriate as an effective biocontrol strategy. These difficulties may also prevent many paratransgenic systems from being appropriately scaled-up to be effective for environmental deployment. Generally, biocontrol means utilizing disease-suppressive microorganisms to eliminate, control or prevent infection, expression and/or transmission of selected pathogens.

The term“nucleic acid” as used herein, refers to a polymer of ribonucleotides or deoxyribonucleotides. Typically,“nucleic acid or“nucleic acid agent” polymers occur in either single or double-stranded form, but are also known to form structures comprising three or more strands. The term“nucleic acid” includes naturally occurring nucleic acid polymers as well as nucleic acids comprising known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Exemplary analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). “DNA”, “RNA”, “polynucleotides”, “polynucleotide sequence”, “oligonucleotide”, “nucleotide”, “nucleic acid”, “nucleic acid molecule”, “nucleic acid sequence”, “nucleic acid fragment”, and “isolated nucleic acid fragment” are used interchangeably herein.

The terms “genetically modified,” “bio-transformed,” “transgenic”, “transformed”, “transformation”, and“transfection” are similar in meaning to“recombinant”.“Transformation”, “transgenic”, and“transfection” refer to the transfer of a polynucleotide into the genome of a host organism or into a cell. Such a transfer of polynucleotides can result in genetically stable inheritance of the polynucleotides or in the polynucleotides remaining extra-chromosomally (not integrated into the chromosome of the cell). Genetically stable inheritance may potentially require the transgenic organism or cell to be subjected for a period of time to one or more conditions which require the transcription of some or all of the transferred polynucleotide in order for the transgenic organism or cell to live and/or grow. Polynucleotides that are transformed into a cell but are not integrated into the host's chromosome remain as an expression vector within the cell. One may need to grow the cell under certain growth or environmental conditions in order for the expression vector to remain in the cell or the cell's progeny. Further, for expression to occur the organism or cell may need to be kept under certain conditions. Host organisms or cells containing the recombinant polynucleotide can be referred to as“transgenic” or“transformed” organisms or cells or simply as“transformants”, as well as recombinant organisms or cells.

A genetically altered organism is any organism with any change to its genetic material, whether in the nucleus or cytoplasm (organelle). As such, a genetically altered organism can be a recombinant or transformed organism. A genetically altered organism can also be an organism that was subjected to one or more mutagens or the progeny of an organism that was subjected to one or more mutagens and has changes in its DNA caused by the one or more mutagens, as compared to the wild-type organism (i.e, organism not subjected to the mutagens). Also, an organism that has been bred to incorporate a mutation into its genetic material is a genetically altered organism.

The term“vector” refers to some means by which DNA, RNA, a protein, or polypeptide can be introduced into a host. The polynucleotides, protein, and polypeptide which are to be introduced into a host can be therapeutic or prophylactic in nature; can encode or be an antigen; can be regulatory in nature; etc. There are various types of vectors including virus, plasmid, bacteriophages, cosmids, and bacteria. Vectors may be stably integrated into a host’s genome as well.

An“expression vector” is a nucleic acid capable of replicating in a selected host cell or organism. An expression vector can replicate as an autonomous structure, or alternatively can integrate, in whole or in part, into the host cell chromosomes or the nucleic acids of an organelle, or it is used as a shuttle for delivering foreign DNA to cells, and thus replicate along with the host cell genome. Thus, expression vectors are polynucleotides capable of replicating in a selected host cell, organelle, or organism, e.g., a plasmid, virus, artificial chromosome, nucleic acid fragment, and for which certain genes on the expression vector (including genes of interest) are transcribed and translated into a polypeptide or protein within the cell, organelle or organism; or any suitable construct known in the art, which comprises an“expression cassette”. In contrast, as described in the examples herein, a“cassette” is a polynucleotide containing a section of an expression vector of this invention. The use of the cassette assists in the assembly of the expression vectors. An expression vector is a replicon, such as plasmid, phage, virus, chimeric virus, or cosmid, and which contains the desired polynucleotide sequence operably linked to the expression control sequence(s). Expression vectors may be stably integrate into a host’s genome as well.

A polynucleotide sequence is“operably linked to an expression control sequence(s)” (e.g., a promoter and, optionally, an enhancer) when the expression control sequence controls and regulates the transcription and/or translation of that polynucleotide sequence. As used herein, the phrase“gene product” refers to an RNA molecule or a protein.

This invention utilizes routine techniques in the field of molecular biology. Basic texts disclosing the general methods of use in this invention include Green and Sambrook, 4th ed. 2012, Cold Spring Harbor Laboratory; Kriegler, Gene Transfer and Expression: A Laboratory Manual (1993); and Ausubel et al., eds., Current Protocols in Molecular Biology, l994-current, John Wiley & Sons. Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology maybe found in e.g., Benjamin Lewin, Genes IX, published by Oxford University Press, 2007 (ISBN 0763740632); Krebs, et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0- 632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569- 8)·

According to a specific embodiment, homolog sequences are at least 60%, 65 %, 70 %, 75%, 80%, 85%, 90%, 95 % or even identical to the sequences (nucleic acid or amino acid sequences) provided herein. Homologs of the sequences provided herein of between 50%-99% may be included in certain embodiments of the present invention.

The nucleic acid agent can be further comprised within a nucleic acid construct comprising additional regulatory elements. For example, transcription from an expression cassette, a regulatory region (e.g., promoter, enhancer, silencer, leader, intron and polyadenylation) may be used to modulate the transcription of the RNA strand (or strands). Therefore, in one embodiment, there is provided a nucleic acid construct comprising the nucleic acid agent. The nucleic acid construct can have polynucleotide sequences constructed to facilitate transcription of the RNA molecules of the present invention operably linked to one or more promoter sequences functional in a mosquito cell. The polynucleotide sequences may be placed under the control of an endogenous promoter normally present in the mosquito genome. The polynucleotide sequences of the present invention, under the control of an operably linked promoter sequence, may further be flanked by additional sequences that advantageously effect its transcription and/or the stability of a resulting transcript. Such sequences are generally located upstream of the promoter and/or downstream of the 3' end of the expression construct. The term “operably linked,” as used in reference to a regulatory sequence and a structural nucleotide sequence, means that the regulatory sequence causes regulated expression of the linked structural nucleotide sequence.

In a further embodiment, a composition including a genetically modified bacteria configured to express an effector peptide or an polynucleotide may be formulated as a water dispersible granule or powder that may further be configured to be dispersed into the environment. In yet a further embodiment, the compositions of the present invention may also comprise a wettable powder, spray, emulsion, colloid, aqueous or organic solution, dust, pellet, or colloidal concentrate. Dry forms of the compositions may be formulated to dissolve immediately upon wetting, or alternatively, dissolve in a controlled-release, sustained-release, or other time-dependent manner. Alternatively or additionally, the composition may comprise an aqueous solution. Such aqueous solutions or suspensions may be provided as a concentrated stock solution which is diluted prior to application, or alternatively, as a diluted solution ready- to-apply. Such compositions may be formulated in a variety of ways. They may be employed as wettable powders, granules or dusts, by mixing with various inert materials, such as inorganic minerals (silicone or silicon derivatives, phyllosilicates, carbonates, sulfates, phosphates, and the like) or botanical materials (powdered corncobs, rice hulls, walnut shells, and the like). The formulations or compositions containing paratransgenic bacteria may include spreader- sticker adjuvants, stabilizing agents, other pesticidal additives, or surfactants. Liquid formulations may be employed as foams, suspensions, emulsifiable concentrates, or the like. The ingredients may include Theological agents, surfactants, emulsifiers, dispersants, or polymers.

As mentioned, an effector peptide or polynucleotide of the invention may be administered as a naked peptide. Alternatively, the peptide of the invention may be conjugated to a carrier known to one of skill in the art, such as a transfection agent e.g. PEI or chitosan or a protein/lipid carrier or coupled to nanoparticles. The compositions may be formulated prior to administration in an appropriate means such as lyophilized, freeze-dried, microencapsulated, desiccated, or in an aqueous carrier, medium or suitable diluent, such as saline or other buffer. Suitable agricultural carriers can be solid, semi-solid or liquid and are well known in the art. Such compositions may be considered“agriculturally-acceptable carriers”, which may covers all adjuvants, e.g., inert components, dispersants, surfactants, tackifiers, binders, etc. that are ordinarily used in pesticide formulation technology.

As mentioned, the nucleic acid agents can be delivered to the mosquito larva in various ways. Thus, administration of the composition to the mosquito larva may be carried out using any suitable or desired manual or mechanical technique for application of a composition comprising a nucleic acid agent, including, but not limited to, spraying, soaking, brushing, dressing, dripping, and coating, spreading, applying as small droplets, a mist or an aerosol. According to one embodiment, the composition is administered to the larvae by soaking or by spraying.

As used herein, the term“gene” or“polynucleotide” refers to a single nucleotide or a polymer of nucleic acid residues of any length. The polynucleotide may contain deoxyribonucleotides, ribonucleotides, and/or their analogs, and may be double-stranded or single stranded. A polynucleotide can comprise modified nucleic acids (e.g., methylated), nucleic acid analogs or non-naturally occurring nucleic acids and can be interrupted by non-nucleic acid residues. For example, a polynucleotide includes a gene, a gene fragment, cDNA, isolated DNA, mRNA, tRNA, rRNA, isolated RNA of any sequence, recombinant polynucleotides, primers, probes, plasmids, and vectors. Included within the definition, are nucleic acid polymers that have been modified, whether naturally or by intervention.

Peptides and proteins for use within the methods and compositions of the invention thus include native or“wild-type” peptides and proteins and naturally occurring variants of these molecules, e.g., naturally occurring allelic variants and mutant proteins. Also included are synthetic, e.g., chemically or recombinantly engineered, peptides and proteins, as well as peptide and protein“analogs” and chemically modified derivatives, fragments, conjugates, and polymers of naturally occurring peptides and proteins. As used herein, the term peptide or protein“analog” is meant to include modified peptides and proteins incorporating one or more amino acid substitutions, insertions, rearrangements or deletions as compared to a native amino acid sequence of a selected peptide or protein, or of a binding domain, fragment, immunogenic epitope, or structural motif, of a selected peptide or protein. Peptide and protein analogs thus modified exhibit substantially conserved biological activity comparable to that of a corresponding native peptide or protein, which means activity (e.g., specific binding to a peptide YY protein, or to a cell expressing such a protein, specific ligand or receptor binding activity, etc.) levels of at least 50%, typically at least 75%, often 85%-95% or greater, compared to activity levels of a corresponding native protein or peptide.

As used herein the terms“approximately” or“about” refer to ± 10%. Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases“ranging/ranges between” a first indicate number and a second indicate number and“ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals there between.

The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean“including but not limited to”. The term“consisting of means“including and limited to”. The term“consisting essentially of’ means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

As used herein, the singular form“a”,“an” and“the” include plural references, unless the context clearly dictates otherwise. For example, the term“a compound” or“at least one compound” may include a plurality of compounds, including mixtures thereof.

It should be noted that for all sequence provided herein, such sequences include all corresponding sequences, so an amino acid sequence include its corresponding DNA or RNA sequence as generally understood by one of ordinary skill in the art.

The invention may be more readily understood by reference to the following examples, which are included merely for the purposes of illustration of certain aspects of the embodiments of the present invention. The examples are not intended to limit the invention, as one of skill in the art would recognize from the above teachings and the following examples that other techniques and methods can satisfy the claims and can be employed without departing from the scope of the claimed invention. Indeed, while this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

EXAMPLES

Example 1 : Delivery of small signaling peptide to control the desire to feed.

Inject small peptides to control the desire to feed: The six processed peptides in the table below were synthesized by GenScript and diluted to 10 ng/ul. Adult female mosquitoes from the MLM colony from south Texas were injected with 1 ng of each of the six peptides. After recovering from the injection, the mosquitoes were starved overnight and the desire to take a bloodmeal was measured the next day (Figure 1). Only three of the synthetic peptides showed a phenotype. Data for the other 3 not shown. Table 4: Full-length protein and cleaved peptide derived from it

Export of small peptide after stable integration into Enterobacter. One exemplary process peptide sequence that demonstrated a strong likelihood to work PFFVGSRY (SEQ ID NO. 20) and was cloned into an 0X19 backbone from Oxford genetics. The protein coding sequence for the small peptide was under the control of the OX19 promoter and downstream of an OmpA secretion peptide. The cassette containing the 0X19 promoter, OmpA sequence (SEQ ID NO. 9), and PFFVGSRY (SEQ ID NO. 20) coding sequence was transferred to the Tn7 plasmid backbone (Figure 2) and transformed into Rho3 cells followed by stable integration into the Ae073 Enterobacter kobei symbiotic bacteria (Figure 3). Expression of the small peptide PFFVGSRY (SEQ ID NO. 20) was measured in the bacterial cell pellet and in outer membrane vesicles (OMVs) by mass spectrophotometry. The small signaling peptide was detected in both the cell pellet and in the OMVs indicated the peptide is efficiently being exported from the symbiotic bacterial cell.

Bacterial delivery of small peptides to control the desire to feed: To determine if bacterial delivery of the small signaling peptide would result in a reduced desire to take a bloodmeal, larvae from the MLM colony of Ae. aegypti were reared in water inoculated with Ae073 Enterobacter exporting the PFFVGSRY (SEQ ID NO. 20) peptide. Mosquitoes were reared to adults and 5-7 day old adult females were starved overnight and the offered a bloodmeal. The percentage of bloodied females was analyzed compared to the number of bloodfed females from the control group that were inoculated with wildtype Ae073 Enterobacter (Data not shown). The present inventors did not observe a difference in the percentage of bloodfed females in the treatment compared to the control. These data supported concluded that the mosquito needs to process the full-length protein into the small cleaved peptides in order to observe a phenotypic effect. As a result, additional directions were directed on delivering the full-length protein for processing.

Example 2: Delivery of full-length protein to mosquitoes to be processed into signaling molecules that control the desire of the mosquito to take a bloodmeal.

Cloning and validation of bacterial constructs: Codon-optimized sequences for E.coli of the full-length proteins (Table 4) (i.e. AAEL011702A (SEQ ID NO. 2), FMRFa (SEQ ID NO. 4), Leucokinin (SEQ ID NO. 6), and MIP (SEQ ID NO. 8)), were cloned into the multiple cloning site of the OX1 backbone from Oxford genetics. The cassette containing the OX1 promotor and the protein coding sequence was transferred into the Tn7 backbone (Figure 5a, 5b, 5c, 5d) and then stably transformed into the Ae073 Enterobacter symbiotic bacteria (Figure 6-9). Expression of the proteins within the bacteria was detected by Western blot (see e.g. Figure 10), indicating we are able to express protein from the Enterobacter symbiont.

Measuring a change in the mosquito’s desire to take a bloodmeal: Aedes aegypti larvae from the MLM colony of mosquitoes collected in south Texas were reared in water inoculated with Ae073 Enterobacter stably integrated with each of the four full-length proteins: AAEL011702A (SEQ ID NO. 2), FMRFa (SEQ ID NO. 4), Leucokinin (SEQ ID NO. 6), and MIP (SEQ ID NO. 8). Mosquitoes were reared to adults and 5-7 day old adult females were starved overnight and the offered a bloodmeal. The percentage of bloodied females was analyzed compared to the number of bloodfed females from the control group that were inoculated with wildtype Ae073 Enterobacter (Figure 11). In one embodiment, when the AAEL011702A protein (SEQ ID NO. 2) is delivered the mosquito, a consistent reduction in the desire to take a bloodmeal was observed. The present inventors do not see a reduction in the desire to take a bloodmeal after delivery of MIP, however, in another embodiment a smaller reduction in the desire to take a bloodmeal after delivery of Leucokinin. In the experiment where mosquitoes were fed bacteria delivering the FMRFa protein a reduction in blood feeding behavior was observed in a replicate group.

The present inventors performed an experiment where we mixed all four bacteria and fed them all together to the larvae. When the mosquitoes are inoculated with all four full-length proteins we see a statistically significant reduction in their desire to take a bloodmeal. This data is comprised of 3 biological replicates. Detection of delivered protein or peptide in the mosquito: To determine if the adult females that did not bloodied contained the protein of interest, the present inventors attempted to detect the protein by Western blot and Mass spectrophotometry. After multiple failed attempts to detect the full-length protein in the mosquito by Western blot and Mass spectrophotometry we hypothesize that we are unable to detect the protein because it is getting cleaved into the small peptides once it is delivered to the mosquito. Notably, however, the present inventors were able to identify AAEL011702A in a protein extract made from 3 pooled mosquitoes by mass spectrophotometry confirming the hypothesis. (Figure 23 and Figure 24)

Protein extracts from adult females that did not take a bloodmeal after being inoculated with the Enterobacter delivering the full-length protein of interest were analyzed on the Mass spectrophotometer to determine if we could detect the correct small peptide originating from full-length protein of interest (Figure 13 and Figure 14). Indeed, the present inventors were able to detect the correct cleavage product in the protein extract from the mosquito which strengthens the argument that the observed reduction in blood feeding behavior was a result of the delivery of the full-length protein and subsequent cleavage into the small signaling peptides.

Example 3: Increase the effect of the desire to take a bloodmeal by including a TAT secretion peptide on the full-length protein.

Cloning constructs that contain a TAT secretion peptide and validating protein expression: In order to assess if the addition of a TAT secretion peptide would increase the reduction in the desire to bloodied, the present inventor cloned the smallest and the largest full- length proteins with and without a TorA (SEQ ID NO. 11) TAT secretion peptide into an OX11 or OX1 backbone respectively and transformed into HT27 E.coli cells (Figure 5a, Figure 5b, Figure 15). Protein expression was measured in the bacteria cells by Western blot. The present inventors are able to detect the full-length proteins FMRFa (SEQ ID NO. 4) and AAEL011702 (SEQ ID NO. 2) in the under the OX11 promotor and with the TAT secretion peptide (Figure 16). We are only able to detect MIP, Leucokinin, and FMRFa in HT27 cells with no TAT secretion peptide and under the 0X1 promoter (Figure 17 and Figure 18).

Detection of delivered protein or peptide in the mosquito: To determine if the mosquitoes fed on bacteria delivering the full-length proteins in fact contained the delivered protein of interest, a Western blot was performed on protein extract from adult females 7 days after bacterial feed. Exemplary protein FMRFa (SEQ ID NO. 4) under the 0X11 promoter and with the TorA secretion peptide (SEQ ID NO. 11) in the mosquito. (Figure 19)

Detecting the cleaved processed peptide in the mosquito:

To determine if the delivered protein was being processed into the appropriate small peptides in the mosquito, mosquito extracts from pools of 3 mosquitoes who ingested HT27 E. coli cells as adults delivering the FMRFa protein with and without a TorA secretion peptide were analyzed by mass spectrophotometry. As shown in Figure 28, the present inventors detected the processed peptides and observed an increase in the relative abundance of the peptide products in mosquitoes fed the construct delivering FMFA with TorA compared to mosquitoes who ingested a construct delivering FMRFa without TorA.

Measuring a change in the mosquito’s desire to take a bloodmeal: Adult females from the MLM colony of mosquitoes were fed a bacterial feeding solution containing the bacteria of interest or a feeding solution with no bacteria. The mosquitoes were allowed access to the bacterial feeding solution for 24 hours and then allowed to sugar feed for 5-6 days. The mosquitoes were starved for 24 hours and then offered a bloodmeal. The percentage of mosquitoes that took a bloodmeal was compared between mosquitoes that had fed on bacteria delivering the protein with a TAT secretion peptide or without a TAT secretion peptide. In one embodiment, the present inventors observed a reduction in blood feeding behavior of mosquitoes fed bacteria delivering the MIP protein (Figure 20). In addition to seeing a reduction in blood feeding behavior in this replicate, the present inventors were able to detect the full-length protein (Figure 25 and Figure 26), and the cleaved product of MIP in the mosquito by Mass Spectrometry (Figure 21).

The present inventors further see an enhancement in the decreased desire to take a bloodmeal in the mosquitoes fed on bacteria delivering the FMRFa with a TAT secretion peptide compared those fed on bacteria delivering FMRFa without a TAT secretion peptide (Figure 22). This data makes sense given the TAT secretion peptides work well to export larger folded proteins. The FMRFa protein is larger than the AAEL011702, sometimes referred to as the AAEL protein.

Example 4: Material and Methods.

Cloning of full-length blood-feeding proteins. Q5 High-Fidelity DNA Polymerase from New England Biolabs (NEB) was used for all cloning, with the following Polymerase Chain Reaction (PCR) thermocycler conditions: 98°C for 30 seconds, followed by 35 cycles of 98°C for 5 seconds, variable annealing temperatures (see table 1) for 30 seconds, and 72°C with variable extension times (see below), and a final extension at 72°C. Products were held at 4°C short term, until used.

An OX19 vector with an OX19 promoter and kanamycin resistance was used as a backbone for the small peptide, PFFVGSRY. The backbone was amplified using the primers, pep PFF For and pep PFF OmpA rev (see primer table 5 below), which contained hanging tails that added the small peptide region to the backbone. The PCR product was ligated using the KLD Enzyme Mix Reaction Protocol (NEB), after which it was transformed into chemically competent E. coli DH5a cells (Invitrogen). Positive colonies were grown in Luria Bertani (LB) medium containing kanamycin, plasmids were isolated using the E.Z.N.A Plasmid Miniprep I kit (Omega Bio-Tek), verified using Sanger sequencing (GENEWIZ, NJ) and CutSmart restriction enzymes (NEB), then used as a template for the TN7 cloning (see below).

An OX1 vector containing a flag-tag, OX1 promoter, and kanamycin resistance was used as a backbone for all the full-length proteins, AAEL011702A, FMRFa, Leucokinin, and MIP, and amplified using the primers, pOX OmpAHA GB Fwd and OX MCS GB Rev (see primer table 5 below for sequences). The inserts were amplified using the following for each construct: AAEL011702A with AELO GB For and AAELO GB rev, FMRFa with FMRF a GB for and FMRFa GB rev Leucokinin, with Leu GB for and Leu GB rev, and MIP with MIP GB for and MIP GB rev (see primer table 5 for sequences, extension times, and annealing temperatures). The NEBuilder HiFi DNA Assembly Master Mix (NEB) was used to assemble each set of the two fragments, and then the constructs were transformed into electrically competent E. coli DH5a cells (Invitrogen). Positive colonies were grown in LB medium containing kanamycin and processed similarly to the PFFVGSRY construct described above.

Cloning of full-length blood-feeding proteins with TAT secretion peptides. An OX11 vector containing a 3xHA tag, an 0X11 promoter, and with kanamycin resistance was used as backbone for the full-length proteins, AAEL011702A and FMRFA, using the primers TAT_GB_for2 and TorA TAT R, annealing at 67°C with an extension time of 1 minute 45 seconds. The AAEL011702A insert was amplified using the primers Aael_Gb_for2 and Aael_GB_rev2 with an annealing temperature of 72°C and an extension time of 30 seconds, and the FMRFa insert was amplified using TAT FMRFA GB F and TAT FMRFA GB R. The NEBuilder HiFi DNA Assembly Master Mix (NEB) was used to assemble each of the two fragments, and then the constructs were transformed into electrically E. coli DH5a cells (Invitrogen). Plasmids were isolated using the E.Z.N.A Plasmid Miniprep I kit (Omega Bio- Tek), then transformed into electrically competent A. coli HT27 cells at 50 ng/ul of plasmid per 50ul of electrically competent cells. Plasmid uptake was verified via sanger sequencing of the plasmid region of interest (GENWIZ, NJ), along with restriction digests using the Cutsmart Buffer and Restriction Endonucleases (NEB). Positive clones were grown in LB with kanamycin and tetracycline, then stored in 25% glycerol stock at -80°C for future use, or fed to adult mosquitos (see below).

TN7 cloning of full-length blood feeding proteins. Regions containing the constructs were amplified using the primers, helper_dsRNA_GB2_for and ds_EGFP_pURR25GB 1 rev, with an annealing temperature of 64°C. A TN7 vector was used as a backbone, which was amplified using pURR25_helper_dsRNA_GB2_rev and pETRR25_GBl_for with an annealing temperature of 63 °C with an extension time of 2 minutes 30 seconds. Constructs were transformed into electrically competent RH03 E. coli and grown in LB containing kanamycin and tetracycline, supplemented with DAP100 at 200mg/ml.

Integration into Enterobacter Ae073. The construct of interest was conjugated into Enterobacter Ae073 using the conjugation protocol for Tn7 integration from the Borlee lab at Colorado State ETniversity as follows. E. coli RH03 with plasmid of interest was grown overnight at 37°C in LB supplemented with 400 ug/ml DAP and kanamycin and carbenicillin at 250 rpm, E. coli RH03 containing the pTNS3 transposase was grown overnight at 37 °C in LB supplemented with DAP and carbenicillin at 250 rpm, and the recipient strain of interest, Enterobacter Ae073 was grown overnight at 30 °C in LB. 400 pL of lOmM MgS0 ₄ was mixed with 100 pL of Enterobacter Ae073, 100 pL of E. coli RH03 with the plasmid of interest, and 400 pL of E. coli RH03 pTNS3. The mixture was centrifuged at 7000 x g for 2 minutes, the supernatant was carefully removed as to not disturb the pellet, and the cells were gently resuspended in 1 mL of sterile 10 mM MgS0 ₄ by pipetting. The suspension was centrifuged at 7000 x g for 2 minutes, the supernatant was removed, and the pellet was resuspended in 30 pL of 10 mM MgS0 _4. The entire conjugation was pipetted onto a sterile cellulose acetate membrane (l3-mm diameter, 0.45- pm pore size) on a pre-warmed LB agar plate containing DAP 400 and incubated overnight at 30°C. A disposable inoculating loop was used to remove the conjugation from the membrane filter and the conjugation was washed by swirling in a microcentrifuge tube containing 1 mL LB medium. The conjugation was centrifuged at 7000 x g for 2 minutes, the supernatant was carefully removed, and this was step was repeated with 1 mL LB medium. The pellet was resuspended in 300 pL LB medium, and plated in various dilutions on LB plates with kanamycin, and incubated overnight at 30°C. Individual colonies were selected and re-streaked to isolate colonies on an LB plate with kanamycin and incubated overnight at 30°C. Individual colonies from the streak plates were verified for integration. Integration was verified by colony PCR using ClonelD lx Colony PCR Master Mix (Lucigen) with the following thermocycling conditions: 98°C for 2 minutes, then 35 cycles of 98°C for 15 seconds, 56°C for 15 seconds, and 72°C for 15 seconds, with a final extension time of 10 minutes. The primers used were Entero glms and pTN7R (see primer table 5 below). Positive clones were grown up in LB liquid culture containing kanamycin at 30°C and stored as 25% glycerol stocks at -80°C.

Larval Feed. Approximately 1000 Ae. aegypti MLM F7 eggs were hatched per 3 liters of sterile water and fed 5 ml of a sterile 10% liver powder suspension. On day 3 post-hatching, larvae were split into sterile cups, at 100 larvae per 150 ml of sterile water. Enterobacter Ae073 containing integrated constructs of interest (grown from glycerol stocks at 30°C for 16-24 hours in LB with kanamycin) were washed in LB to remove any antibiotics, then resuspended in 50 ml of sterile water. These 50 ml resuspensions of bacteria were added to the 150 ml of sterile water containing 100 larvae, bringing the volume up to 200 ml and reaching a final OD600 of 0.6 - 0.7. 400 pL of the sterile 10% liver powder suspension was added to each cup. Pupae were collected daily on days 6-9 post-hatch and placed in cups in small adult mosquito cages (BugDorm) and provided with cotton balls soaked in sterile 10% sucrose solution. Remaining larvae were given 200-400 pL 10% liver powder suspension as needed until most the larvae pupated. On day 13 post-hatch the adult mosquitos were starved for 12-16 hours by removing the 10% sucrose-soaked cotton balls. On day 14 post-hatch, the adult mosquitos were offered a bloodmeal (see below) for 5 minutes and their blood feeding rate was measured as the number of blood-fed females over the total number of females. All female mosquitos were collected and frozen at -80°C for later analysis (see below).

Adult Feed. 35-40 1 week old adult Ae. aegypti MLM female mosquitos were sorted into small adult cages (made of 32 oz wax and cardboard soup cups with mesh lids), and starved overnight. Starved mosquitos were then fed bacteria as follows: HT27 E. coli bacteria containing constructs of interest were grown overnight at 37°C in LB with kanamycin and tetracycline, then washed in fresh LB to remove any antibiotics. Pellets were resuspended in LB broth with no antibiotics and adjusted to have an OD600 of 1.0 +/- 0.07. The cultures were aliquoted into l5ml, then spun down and pelleted. The bacterial pellet was resuspended in l5ml of sterile solution composed of l50mM NaCl, lOmM NaHC03, lmM ATP, 2% sucrose, and 1% liver powder suspension, placed in 2 oz plastic cups covered in nonwoven sterile gauze held in place by a rubber band, and given to the starved adult mosquitos. Adult mosquitos were allowed to feed for 24 hours, after which the bacterial feeding solution was removed and replaced with cotton balls soaked in 10% sucrose solution for approximately 24 hours. Mosquitos were then starved for 16- 24 hours by removing the sucrose source. After starving, the mosquitos were offered a bloodmeal for 5 minutes (see below), and their blood feeding rate was measured as detailed above.

Mosquito Blood-feeding. Mosquitos were offered a bloodmeal using an the Hemotek artificial feeding system. Each feeder was covered with pork sausage casing, and 2 ml of defibrinated calf blood purchased from the Colorado Serum Company was added to each feeder, along with lOOul of 0.5M ATP. Mosquitos were allowed to feed for 15 minutes, after which their blood feeding rate was measured as number of blood-fed female mosquitos over the total number of female mosquitos.

Mosquito rearing. Larvae were reared in pans containing 400 larvae per 6 L of distilled water. Larvae were fed a suspension of 10% liver powder as needed. Pupae were removed daily as they emerged, placed in sterile cups of water, and placed in BugDorm-l Insect Rearing Cages. The adults were maintained in an insectary at 80% humidity and 29° C with a light/dark cycle of 12/12. Adult mosquitos were allowed to feed on 10% sucrose solution provided in 50ml Erlenmeyer flasks with dental cotton dipped into the solution as wicks. For colony maintenance, mosquitos were fed defibrinated calf blood (Colorado Serum Company) using the Hemotek artificial feeding system. Each feeder was covered with pork sausage casing. 2ml of defibrinated calf blood was added to each feeder with lOOul of 0.5M ATP, and mosquitos were allowed to feed for up to an hour. 2-3 days post feeding, sterile 50ml beakers containing a small amount of water and a soaked paper towel were placed in each cage. Egg papers were collected 5-6 days post bloodmeal, and allowed to desiccate in the insectary until hatched. Outer Membrane Vesicle (OMV) Extraction. Outer Membrane vesicles (OMV) were extracted from liquid bacterial cultures using the ExoBacteria™ OMV Isolation Kit (for E.coli and other gram-negative bacteria) (System Biosciences). OMV’s were also extracted using the following method; Bacteria was seeded into 400 mL of LB medium containing the appropriate antibiotics and grown for 48 hours. Cultures were centrifuged in 250 mL bottles at 3750 rpm for 20 minutes at 4°C, after which the supernatant was poured into fresh 250 mL bottles and the spin was repeated. The supernatant was filtered first through a 45 pm vacuum filter, then through a 22-pm filter. The final filtrate was loaded into 100 mL ultrafuge tubes and weighed to perfectly balance the tubes. The filtrate was spun at 36,000 rpm at 4°C for 3.5 hours in a pre-chilled rotor. After spinning, the supernatant was carefully poured out and the OMV pellet was resuspended in 500 pL of B-PER buffer. OMV extractions were saved for Mass Spectrometer analysis.

Example 5: Analytical Methods.

Protein extraction. Collected mosquitos were stored at -80°C. 100 pL of“I-PER” Buffer (0.1% TritonX-lOO, 8M Urea, 25mM Tris-HCl, lOOmM NaCl, at pH7, with Protease Inhibitor cocktail) or RIPA buffer (l50mM NaCl, 5mM EDTA pH8, 50mM Tris pH8, 1% Triton X-100, 0.5% Sodium Deoxycholate, 0.1% SDS, and protease inhibitors) was added to a tube containing a pool of 3 mosquitos each, and ground with a motorized pestle and allowed to incubate for 5 minutes. The mosquito and buffer mixture were then spun down for 5 minutes at the maximum centrifuge speed and the supernatant was carefully removed from the mosquito debris and approximately 500 pL of protein extract was added to an Amicon® Ultra 0.5 mL centrifugal filter (Millipore, Ireland) with a 3,000 MWCO, and then centrifuged (15,000 rpm x 20 min x RT) to separate intact proteins from signaling peptides. The filtrate was stored at -20°C until processed.

Protein quantitation. Protein concentration was approximated using a NanoDrop (Thermo). Lysis buffer was used to blank the instrument, and absorbance values were collected at 280 nm and used to approximate protein concentration (mg/mL).

Protein digestion. Intact protein samples were reduced, alkylated and digested following the protein digestion protocol from the University of Washington Proteomics Resource with a few modifications (UWPR, 2011). Intact protein samples were reduced by addition of 1 M dithiothreitol (DTT) prepared in 0.1 M ammonium bicarbonate to a final concentration of 50 mM, and allowed to incubate for 25 min at 45°C. Samples were cooled and then alkylated by addition of 0.5 M iodoacetamide (IAA) prepared in 0.1 M ammonium bicarbonate to a final concentration of 55 mM and incubation at room temperature in the dark for 15 min. Samples were then diluted 1 :9 with 0.1 M ammonium bicarbonate, and MS grade trypsin (Fisher) was resolubilized in 0.1 M ammonium bicarbonate added at a ratio of 1 : 100 enzyme: substrate. Protein digestion was performed at 37°C on a shaker plate set to 200 rpm overnight, and the reaction was quenched the following day by addition of glacial acetic acid until pH < 2 was achieved. Peptide mixtures were then concentrated and desalted using Cl 8 ZipTip® pipette tips (Millipore).

Peptide desalting. Peptides were desalted prior to analysis by LC-MS to remove salts and SDS from the lysis buffer. A 10 pL ZipTip® packed with 0.6 pL Cl 8 resin was selected for peptide desalting and concentration. Both signaling peptides and peptide mixtures resulting from protein digestions were processed as follows. The ZipTip® was conditioned by aspirating with 10 pL 0.1% formic acid three times. Sample was loaded by aspirating the sample five times, and then washed by aspirating with 10 pL 0.1% formic acid three times. Peptides were eluted by aspirating 50 pL of 80% acetonitrile containing 0.1% formic acid five times. Eluted peptides were dried using a CentriVap benchtop vacuum concentrator (Labconco) operated at room temperature for approximately 20-30 min, and then resolubilized in 50 pL 1% acetonitrile supplemented with 0.1% formic acid and 100 fmol/pL Glu'-fibrinopeptide B as an internal standard (ISTD).

LC-MS. Signaling peptides and peptide mixtures were both analyzed by LC-MS/MS using a Synapt G2-Si high definition mass spectrometer (HDMS) coupled to an ACQUITY M- Class ultra-performance liquid chromatograph (UPLC) with an electrospray ionization (ESI) source (Waters). Capillary voltage for sample and LockSpray sprayers was set to 3.6 and 3.0 kV, respectively. Sampling cone and source offset were both set to 40, and source and desolvation temperatures were set to 100 and 200 °C, respectively. Desolvation gas flow rate was 650 L/hr and nebulizer was set to 6.5 bar. Glu'-fibrinopeptide (Sigma, US) was prepared at a concentration of 100 fmol/pL in acetonitrile:water (1 :1 v/v) containing 0.1% formic acid and used as the LockMass solution. LockSpray measurements (Glu'-fibrinopeptide: 785.8426 m/z) were collected every 60 s for both signaling peptide and peptide mixture analysis methods.

Signaling peptides were separated on an ACQUITY UPLC M-Class HSS T3 Column (100 A pore size, 1.8 pm particle size, 300 pm ID X 100 mm L) (Waters) following a linear gradient using mobile phases (a) water with 0.1% formic acid and (b) acetonitrile with 0.1% formic acid (Table 1). The flow rate was maintained at 5 pL/min, and 1 pL injections were made in duplicate. MS data were collected by high definition data-independent acquisition (HDMSe) in positive polarity (ES+) and resolution (FWHM = 20,000) modes. Data were collected in continuum mode, and LockSpray data were acquired by not applied. The mass range was 50-2000 m/z, and 0.486 s scans were collected with a 0.014 s interscan delay. Default ion mobility settings were used, and a trap collision energy ramp of 12-40 V was applied.

Table 1. LC gradient for signaling peptide separation. Time (min), flow rate (pL/min), % mobile phase A (water + 0.1% formic acid) and B (acetonitrile + 0.1% formic acid), and gradi ent curve ( 1 - 11 ) value s are provi ded .

Peptide mixtures were separated on an ACQUITY UPLC M-Class HSS T3 Column (100 A pore size, 1.8 pm particle size, 300 pm ID X 100 mm L) (Waters) following a linear gradient using mobile phases (a) water with 0.1% formic acid and (b) acetonitrile with 0.1% formic acid (Table 2). The flow rate was maintained at 5 pL/min, and 1 pL injections were made in duplicate. Data were collected by HDMSe in positive polarity (ES+) and high resolution (FWHM = 40,000) modes. Default ion mobility settings were used, and a collision energy ramp of 12-40V was used. Data were acquired in continuum mode, and LockSpray data were collected but not applied during acquisition. The mass range was 300-2000 m/z, and scan time was set to 0.486 s with a 0.014 s interscan delay.

Table 2. LC gradient for separation of peptide mixtures. Time (min), flow rate (pL/min), % mobile phase A (water + 0.1% formic acid) and B (acetonitrile + 0.1% formic acid), and gradient curve (1-11) values are provided.

Signaling peptide identification. Analytical standards for the six signaling peptides were synthesized by ## (##). Peptide standards prepared at 0.1 ng/mL containing 100 fmol/pL Glu'-fibrinopeptide as an ISTD were used for method development, to determine retention time and mass error (Table 3, Figures 1-6). Additionally, peptide standards were used for relative quantitation of signaling peptides recovered from mosquito protein extracts. Glu'-fibrinopeptide was used as an internal standard to calculate the calibration response factor for the six signaling peptides. Additionally, Glu'-fibrinopeptide was used as the Lock Mass solution for accurate mass measurement (AMM) of the signaling peptides. The AMM correction was applied in MassLynx, and then peak area and relative quantitation data were processed in QuanLynx and Excel, respectively.

Table 3. Signaling peptide molecular formula, isoelectric points (pi), mass error (ppm), observed mass-to-charge ratio (m/z), expected m/z, and retention time (min) information.

Figure 1. Extracted ion chromatogram for PFF signaling peptide (486.7507 m/z) observed at 12.95 min; Figure 2. Extracted ion chromatogram for SAL signaling peptide (410.2097 m/z) observed at 13.87 min; Figure 3. Extracted ion chromatogram for AGQ signaling peptide (457.2112 m/z) observed at 12.86 min; Figure 4. Extracted ion chromatogram for SDP signaling peptide (551.8221 m/z) observed at 13.34 min; Figure 5. Extracted ion chromatogram for TWK signaling peptide (545.2775 m/z) observed at 13.36 min.; Figure 6. Extracted ion chromatogram for NNP signaling peptide (604.2802 m/z) observed at 14.87 min.

Protein identification. All peptide data were processed using Progenesis QI for proteomics (Nonlinear). Peptide spectra were searched against a custom database generated from ##protein sequences from SnapGene constructs##. The MSe identification workflow was used with default settings for Apex3D parameters, auto-alignment and peak picking. Alignment of peptide samples was conducted automatically using the most suitable fit. Tolerance parameters and ion matching requirements for protein identification were set as follows: 10.0 ppm mass error for peptide ions, 3 fragments per peptide for peptide ion identification, 4% false discovery rate (FDR) and 2 peptides per protein for protein identification. Abundance of both non-conflicting and unique peptides was used for relative quantitation of identified proteins. Abundance values for identified proteins were exported into Excel as .csv files, and were standardized across samples using estimated protein concentration.

Table 5. List of Primers.

Table 6. Mass spectrophotometry showing small peptide export from Enterobacter .

Table 7a. Mass Spec detection of processed peptide after feeding the full-length protein.

Table 7b. Mass Spec detection of processed peptide after feeding the full-length protein.

REFERENCES

The following references are hereby incorporated in their entirety by reference:

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SEQ ID NO.1

DNA

AAEL011702A

Aedes aegypti

ATGAAGTTAATCGCGCGTAGTCACCACCCAGCAACAGTCTTATGTTCTGCATCCTCGCTT ATTTTATTAG TGTCCGCCGTCTTAGTGATGTCGTCCATTATGTGTGGGGCGGACCCTTCTCCATTAGAGT CTAATTCGTT GTTCGGGGGAGTAGACGAGAAAAACCTGAACGATAAGCGTCCTTTTTTCGTAGGTTCCCG TTACGGTCGT AGTCACGTCTACGGCGGGAAAGACCTGCGTCAAGTCAATGTCGTGCCTCGCAATGACCGT TTCTTCCTGG GATCACGCTACGGCAAACGTAGTGACACGCTTACCAAAGAAATTGAAACGGACAACAACA ACGGGGCAGA GCTGACTTATCTTGCTTGCCTTCATACTGGAGTGTCGAATCTGTATCGTTGCTATGGGAA AGAGCGTGAC C AGC AAT AC AAC GAGG AT T T GG AC AGT T CC T C T C C AT AA

SEQ ID NO.2

Amino Acid

AAEL011702A

Aedes aegypti

MKLIARSHHPATVLCSASSLILLVSAVLVMSS IMCGADPSPLESNSLFGGVDEKNLNDKRPFFV GSRYGRSHVYGGKDLRQVNWPRNDRFFLGSRYGKRSDTLTKEIETDNNNGAELTYLACLH TGV SNLYRCYGKERDQQYNEDLDSSSP

SEQ ID NO.3

DNA

FMRFa

Aedes aegypti

ATGAAGATGTATCTTTTTTTAGCCATTCTGATCTATAAGTGTTCCAATTATTTCAGT TGTGCGG AGTACGAATTAGCTGAGGCAAGCCAGGCAGTGAATGGTCCTTCTATTGCATTAGACCTTA ACCC GGAAAGCGAAAATGACTTTACCAACAACAATAACGGCATGGTCTTTGAGTTTCGCCGTTC GATT AAATCAGATAACACGGAAATTGAGGCCCGCCGTCGTTCAGCTCTGGACAAAAACTTCATG CGTT TTGGACGCACCGACCCTACAGCTTTACAGCGCGTGGCTCGTGCAAGCAAACAGGCTAATT TGAT GCGTTTCGGACGCGCAGGCCAAGGGTTTATGCGCTTTGGGCGTATCCCGGAGAATGTCCC CCTT TCTTTGGTTCATAACGATGACGACTCTCGTCAGGATTACAACGGGGACTCGGAGGAGTCC AGTG AAGAGAC AAC T T AC T C CAAAC G C AC T C C GAAC T C T GAC GAGAT C AC G GAG G T T T C T GAAT C C G G TGAACAAATTAAACCCAAGCAATTGGTTTACTACCGCCGTGACTCACCCAAAAATTTAAT GCGC TTCGGCAAGCGTGATGACACCAATAAATTTTTGCGTTTGAGTCGTGCAAATCTGATGCGC TTTG GACGCGCAGGTTCCGAAGCTGGGGGCAACCTTCAGCGCACAAATTTTCTGCGTTTCGGTC GTGG TAGTGGTAATCTGATGCGCTTTGGGCGTGCTAAAGGCAATCTTATGCGCTTTGGGCGCAG TGAT CCCCGTTTCTTGCGTCTGGTTAAAATGGACAATAACTTTATGCGCTTCGGGCGCTCAGAC AAAG C AC T TAAG T C C G T AGAC AAGAAT GAAAC CCGCTCATCG GAG T C AT C C AC T T C AC AT AAT AAT GA AATTATTAGC GAGAC CAAAC AC CAT GAC GAC T T G T C C T T AGAT AAGAC T GAC G C C C CAT AC AAA T C AGAC GAG GAT TAT GAAAT C C AG T T G C G C GAG GAAGAT AT T T T AAC AC CGGTATTTGTG T AA

SEQ ID NO.4

Amino Acid FMRFa

Aedes aegypti

MKMYLFLAILIYKCSNYFSCAEYELAEASQAVNGPS IALDLNPESENDFTNNNNGMVFEFRRS I KSDNTEIEARRRSALDKNFMRFGRTDPTALQRVARASKQANLMRFGRAGQGFMRFGRIPE NVPL SLVHNDDDSRQDYNGDSEESSEETTYSKRTPNSDEITEVSESGEQIKPKQLVYYRRDSPK NLMR FGKRDDTNKFLRLSRANLMRFGRAGSEAGGNLQRTNFLRFGRGSGNLMRFGRAKGNLMRF GRSD PRFLRLVKMDNNFMRFGRSDKALKSVDKNETRSSESSTSHNNEI ISETKHHDDLSLDKTDAPYK SDEDYEIQLREEDILTPVFV

SEQ ID NO. 5

DNA

Leucokinin

Aedes aegypti

ATGGCAATGTTGTTACAAGTCGCGCTGCCCCTGTTGGCCGCGGTCTCTTGGGGATGGGAG TTAA ATGAGAACGATGATAGCCTTGCGAAAATTATTGAGGGCTGTGAATGGACATCCCGTCAAA ATGT AATTAGTGAAATTTTGCTGGACCGTTATCGTAAGTATGCCATGTACAACTTCTTCCTTCT TGAT GACGTATGCGCCGTGCATGAGTGGAATAAAAATTTAAAAGAACCAGAGTTTTCAGAGAAT AACG AGGCAGAAGACAAGTCGCCTACGAGCGCTCAGAACACTCAAGAGCATATCCCTGGAAATA ACTT CCCTCCACCCGCGGCAAGCAATCCTCCGGTGAACAGTAGCTGCGCTAAGAGTGCGAAAGA CTTC TTTATCTGCTTATCTAATCAGCTTGGCGATCCAACGCTGAACGCCATGTTGTTGGATAAT CTTG AGGTGGCTTGCGACCCACGCTTCTCCCCAGTATCGGCTATCCAAAAACGTAATAGCAAAT ACGT GTCCAAACAGAAGTTCTATTCTTGGGGTGGGAAGCGTAATAATCCAAACGTGTTTTATCC CTGG GGTGGGAAACGCAACACTGGACGTGTACATCGTCAGCCAAAAGTTGTAATCCGTAATCCT TTCC ATGCGTGGGGCGGCAAGCGTAACCAAAAGGATGACAATGTTTTCTAA

SEQ ID NO. 6

Amino Acid

Leuckokinin

Aedes aegypti

MAMLLQVALPLLAAVSWGWELNENDDSLAKI IEGCEWTSRQNVISEILLDRYRKYAMYNFFLLD DVCAVHEWNKNLKEPEFSENNEAEDKSPTSAQNTQEHIPGNNFPPPAASNPPVNSSCAKS AKDF FICLSNQLGDPTLNAMLLDNLEVACDPRFSPVSAIQKRNSKYVSKQKFYSWGGKRNNPNV FYPW GGKRNTGRVHRQPKWIRNPFHAWGGKRNQKDDNVF

SEQ ID NO. 7

DNA

MIP

Aedes aegypti

ATGATCAATCAACATCTTATCCTGTGGACTAACTTTGGTAAACTGCTGTTACTTCTTGTC CTGT GCTCTCTGGTCTCAAATATTCAAACTGAATCGGCTTCCCTGGAACAACATCAGATGGAAC ATGC GGATGAGTCGACACACTCTCATTCCCCTCAAAAGCGCACATGGAAGAACTTGCAAGGTGG CTGG GGTAAACGTACGCCGACAAGTGAGCAACCCGACCCCAACGCAGACTATTACGGCTATACA GGAC GTAATGACGACACCGCCGACTATGGTAACGCAGAAAATGAGTTAGACAAATTAAATAAAT ATTT GATCAAAGGACTTATTAACCAACGCTTGGCGCAGCTTGATACTCAATACGATGGATCCGA CGAA GAGTACCCAGTCGAGAAACGTGCGTGGAACAAGATCAACGGGGGATGGGGTAAGCGTGTC AACG CCGGACCCGCACAGTGGAATAAGTTTCGCGGATCATGGGGCAAACGTGAACCAGGGTGGA ATAA TCTTAAGGGTCTGTGGGGCAAGCGTTCTGAAAAGTGGAACAAATTGAGCTCATCATGGGG AAAA CGCGACAGCGGGAATAGCAACAGCTATTAA

SEQ ID NO. 8

Amino Acid

MIP

Aedes aegypti

MINQHLILWTNFGKLLLLLVLCSLVSNIQTESASLEQHQMEHADESTHSHSPQKRTWKNL QGGW

GKRTPTSEQPDPNADYYGYTGRNDDTADYGNAENELDKLNKYLIKGLINQRLAQLDT QYDGSDE

EYPVEKRAWNKINGGWGKRVNAGPAQWNKFRGSWGKREPGWNNLKGLWGKRSEKWNK LSSSWGK

RDSGNSNSY

SEQ ID NO. 9

DNA

OmpA

E. Coli

ATGAAAAAGACGGCGATTGCTATCGCTGTGGCGCTTGCTGGATTCGCCACTGTAGCACAA GCA

SEQ ID NO. 10

Amino Acid

OmpA

E. Coli

MYHSLGTPGFTLYASGSYNVWCWLALAIDLCRSYHPLDTPASGRCQASELSNSKEVPTMK KTAI AIAVALAGFATVAQA

SEQ ID NO. 11

Amino Acid

Tor A

E. Coli

ATGAACAACAACGACCTTTTTCAGGCTTCTCGCCGTCGCTTTTTGGCGCAACTTGGGGGC TTGA

CGGTGGCGGGAATGCTTGGACCCTCCTTACTTACGCCTCGCAGAGCCACAGCT

SEQ ID NO. 12

Amino Acid

Tor A

E. Coli

MNNNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATA

SEQ ID NO. 13 Amino Acid

NPYLR7

Aedes aegypti

MNFTAEFFPENGTRPSEWATFDCDSSSYIPQGIASLRFQILIYLAYSAIFLTSIVGNVAV FLWHLLPRMNTVTNL FIANLALGDMLMTI FCI PFSFVSI FVLQYWPFGAI ICRIVNYSQAI SVLVSAYTMIAI SADRYLAIMWPLKPRVTKR LARILILLVWTGALATAAPIPVFSTLIQPTEFYDYCDLSICTEVWPDDHSDHGYSLTLMT LQFLAPLIVLITTYVQI ACKVWAKTPPGESVKQRDRRILQSKRKMIKMMITWAVFTICWLPFNIFMLVPLDPDWRPL PYLWFLFHWLAMSHSC YNPIIYCYMNEKFRQGFQRLVEMIGRRCCCWMGATRRKSDASTNTEMLQLFPSTTLMTTV ASGVGSAAGSREWCRKG L

SEQ ID NO. 14

DNA

NPYLR7

Aedes aegypti

ATGAACTTCACTGCCGAGTTCTTCCCGGAAAACGGGACTCGACCATCCGAGGTCAACGCA ACCT TCGACTGCGACAGTTCGTCCTACATCCCGCAAGGAATCGCCTCGCTCCGCTTCCAGATCC TGAT CTACCTGGCGTACAGTGCGATCTTCCTGACCTCGATCGTCGGCAACGTGGCCGTATTCCT CGTG GTCCACCTGCTACCCCGCATGAACACCGTCACCAACCTCTTCATCGCGAACCTCGCCCTT GGTG ACATGCTCATGACCATCTTCTGCATCCCGTTCTCGTTCGTGTCGATCTTCGTCCTGCAGT ACTG GCCCTTCGGCGCGATCATCTGCCGCATCGTTAACTACTCGCAGGCCATCTCGGTGCTGGT CAGT GCCTACACCATGATCGCGATCAGTGCCGATCGCTACCTGGCGATCATGTGGCCCCTCAAA CCCC GGGTCACCAAACGCCTCGCTCGGATCCTCATCCTTCTGGTCTGGACCGGAGCTCTAGCCA CCGC CGCTCCCATCCCTGTCTTCTCCACCCTGATCCAACCGACCGAATTCTACGACTACTGCGA CCTC TCGATCTGCACCGAAGTCTGGCCCGACGACCACTCCGATCACGGCTACTCCCTAACCCTG ATGA CCCTCCAATTCCTGGCCCCACTGATCGTCCTCATCACCACCTACGTCCAGATCGCCTGCA AGGT CTGGGCCAAGACCCCTCCGGGCGAATCCGTCAAACAGCGCGATCGACGCATCCTCCAGTC CAAG CGTAAGATGATCAAGATGATGATCACCGTGGTGGCGGTCTTCACGATCTGCTGGCTGCCG TTCA ACATCTTCATGCTGGTTCCGCTGGACCCCGACTGGCGTCCGCTGCCCTACCTGTGGTTCC TGTT CCACTGGCTGGCGATGTCGCACAGCTGCTACAATCCGATCATCTACTGCTACATGAACGA GAAG TTCCGACAGGGGTTCCAGCGACTGGTGGAGATGATCGGGCGTCGGTGTTGCTGCTGGATG GGCG CGACGCGGAGGAAGAGTGATGCCAGCACCAATACGGAGATGCTGCAGTTGTTTCCGAGCA CGAC GCTGATGACCACGGTGGCCAGTGGCGTTGGGAGTGCTGCGGGGAGTCGCGAGTGGTGCCG GAAG GGGTTGTAG

SEQ ID NO. 15

Amino Acid

FMRFa-l processed peptide

Aedes aegypti

SALDKNFMRF

SEQ ID NO. 16

Amino Acid

FMRFa-3 processed peptide Aedes aegypti

AGQGFMRF

SEQ ID NO. 17

Amino Acid

FMRFa-lO processed peptide

Aedes aegypti

SDPRFLRLV

SEQ ID NO. 18

Amino Acid

MIP-l processed peptide (MIP)

Aedes aegypti

TWKNLQGGW

SEQ ID NO. 19

Amino Acid

Leucokinin-3 processed peptide

Aedes aegypti

NNPNVFYPWG

SEQ ID NO. 20

Amino Acid

AAEL011702A processed peptide (AAEL)

Aedes aegypti

PFFVGSRY

SEQ ID NO. 21

Amino Acid

AAEL-TorA fusion

Artificial

MNNNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATAMKLIARSHHPATVLCSASSLI LLVS AVLVMSSIMCGADPSPLESNSLFGGVDEKNLNDKRPFFVGSRYGRSHVYGGKDLRQVNVV PRND RFFLGSRYGKRSDTLTKEIETDNNNGAELTYLACLHTGVSNLYRCYGKERDQQYNEDLDS SSP