Control of mosquito

Abstract

To provide an ovitrap and a method introducing bovine urine for the control of mosquito populations.SOLUTION: The term bovine urine as used here includes liquid concentrates and solid forms such as powders or tablets, and more preferably includes products derived from bovine urine which are provided in unit dosage form for ease of use. The product may further comprise instructions for dosing at a given larvicidal concentration in an ovitrap filled with a known volume of water.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to products, methods and uses of cow urine for the control of mosquito populations. The term "cow urine" as used herein includes products derived from cow urine, including liquid concentrates and solid forms, such as powders or tablets, more preferably provided in unit dosage form for ease of use. The products may further include instructions for administration at a given concentration. [Background technology]

[0002] As exemplified by Applicant's own International Patent Application PCT / IB2018 / 000965 (WO2019 / 043449; PCT Publication No. 2019 / 023449), the prior art teaches ovitraps, which use attractants, such as chemicals that provide ovipositor cues that attract insects, and alternative means, such as insecticides, including larvicides and adulticides, or mechanical means to kill mosquitoes and / or their larvae.

[0003] Mosquitoes are vectors of many diseases, such as, but not limited to, malaria, dengue fever, chicken guinea, filariasis, yellow fever, Japanese encephalitis, and Zika virus, and thus there is a need to increase the efficacy of traps and related methods for effective control.

[0004] Indeed, current mosquito control programs around the world face challenges arising from the large number of mosquito species, the diversity of their habitats and their contact with humans.

[0005] Vector control strategies aim to modify the behaviour of ovipositing females and disrupt the development of eggs, larvae and pupae, thereby resulting in population reductions, and therefore the use of ovitraps and insecticides has become commonplace.

[0006] The approximately 3,500 species of mosquitoes belonging to 43 genera are grouped into two main subfamilies, Anophelinae and Culicinae.

[0007] This distinction is of great practical importance, as the two subfamilies tend to differ in their importance as vectors of various types of diseases.

[0008] Human malaria is transmitted exclusively by females of the genus Anopheles.

[0009] On the other hand, arboviruses such as yellow fever and dengue fever tend to be transmitted primarily by Culicine species, although not necessarily in the Culex genus.

[0010] The two main groupings within the genus Anopheles are one formed by the subgenera Cellia and Anopheles, and the other formed by Kerteszia, Lophopodomyia and Nyssorhynchus.

[0011] The major species known to carry human malaria are those within the subgenus Anopheles.

[0012] The subfamily Culicinae is divided into 11 families, namely: ·Aedeomyiini; ·Aedini (including Aedes species); ·Culicini (including Culex species); ·Culisetini; ·Ficalbiini; ·Hodgesiini; ·Mansonini; Orthopodomyiini; ·Sabethini; Toxorhynchitini; and ·Uranataeniniini It has 3,046 species in 108 genera that are classified as

[0013] The International Journal of Pharmacy and Pharmaceutical Science, Vol. 6, No. 3, 2014, pp. 20-22 (Non-Patent Document 1), reviews the various uses of cow urine and describes it as an ovipositor cue for Anopheles gambiae and Culex quinquefasciatus.

[0014] Apart from that, the document also describes cow urine as a biopesticide and bioenhancer in agricultural practices.

[0015] Other documents disclose the use of attractants:

[0016] EA 026601 discloses an aerosol containing an attractant.

[0017] Hawaria, Dawat et al., using textile strips soaked in cow urine as an attractant. J Infect Dev Ctries, 2016, 10(1), pp. 082-089 (Non-Patent Document 2).

[0018] Kweka et al., Parasites & Vectors, 2011, 4, 184, looked at the effect of cow urine (fresh and aged) on spawning by filling bowl-shaped containers with paper-lined sides with soil, water and cow urine. These would not be considered ovitraps.

[0019] Kweka et al., Parasites & Vectors, 2010, 3, 75, discuss an odor-based resting box for sampling mosquitoes.

[0020] Also, Mahande AM et al., BMC Infect Dis, 15 Jun 2010, 10, 172 (Non-Patent Document 5), who used urine-soaked cloth in a baiting box.

[0021] Also, Kweka et al., Malaria Journal, 2009, 8, 82 (Non-Patent Document 6), used urine-soaked cloth in a feeding lure box.

[0022] Most importantly, however, it has not previously been recognized that cow urine acts both as a mosquito attractant and (very importantly) as a mosquito larvicide, making it useful as a natural product for controlling mosquitoes independent of additional insecticides. [Prior art documents] [Patent documents]

[0023] [Patent Document 1] International Publication No. 2019 / 043449 [Non-patent literature]

[0024] [Non-Patent Document 1] International Journal of Pharmacy and Pharmaceutical Science, Volume 6, Issue 3, 2014, Pages 20-22 [Non-Patent Document 2] Hawaria, Dawat et al. J Infect Dev Ctries, 2016, 10(1), pp. 082-089 [Non-Patent Document 3] Kweka et al., Parasites & Vectors, 2011, 4, 184 [Non-Patent Document 4] Kweka et al., Parasites & Vectors, 2010, 3, 75 [Non-Patent Document 5] Mahande AM et al., BMC Infect Dis, June 15, 2010, 10, 172 [Non-Patent Document 6] Kweka et al., Malaria Journal, 2009, 8 82 Summary of the Invention [Problem to be solved by the invention]

[0025] It is an object of the present invention to provide a simpler, more effective ovitrap and population control method for use therewith. The fact that cow urine acts as a larvicide allows for the addition of cow urine to the water of the ovitrap in larvae-killing amounts, as opposed to, for example, soaking fabric only to attract mosquitoes to an area. [Means for solving the problem]

[0026] According to a first aspect of the present invention there is provided an ovitrap comprising a container which in use is filled with water, an oviposition surface on which mosquitoes settle and lay eggs in the water, characterised in that it contains a water conditioner which is also larvicidal such that in use the trap does not contain any added insecticide.

[0027] Preferably, the Ovitrap is doped with cow urine as a water conditioner and larvicidal agent. Thus, the Ovitrap may be provided as a kit together with a unit dosage form of cow urine and / or instructions for use including cow urine and advising on its appropriate dosing levels for the Ovitrap.

[0028] Preferably, but not necessarily, the cow urine is from Bos indicus, Bos Taurus or Zebu.

[0029] The composition of cow urine typically includes, in addition to water, 40-60% by weight urea, and 40-60% by weight other components including minerals, salts, hormones and enzymes. See, for example, International Journal of Res Ayurveda pharm 8[5], 2017, pp. 1-6, which is incorporated by reference.

[0030] Biochemical analysis of cow urine has shown that other constituents include elements including sodium, calcium, nitrogen, sulfur, manganese, iron, silicon, chlorine, phosphorus and magnesium, either alone or as minerals or salts, vitamins, acids such as citric acid, uric acid and phenolic acid, as well as sugars such as lactose, proteins and creatinine.

[0031] In particular, the presence of urea, creatinine, gold hydroxide, phenol, calcium and magnesium have been suggested to contribute to the antimicrobial properties of cow urine.

[0032] The enzymes include proteases, chitinases, and lipases that act on mosquito larvae.

[0033] Additionally, microorganisms present in cow urine and / or attracted to the conditioned water aid this process, which is outlined in Figure 1.

[0034] Preferably, the cow urine is provided in a unit dosage form.

[0035] The unit dosage form can be a powder, granules, tablet or liquid with a metered dispenser.

[0036] Applicants further analyzed several different forms of cow urine for phenolic, flavonoid and amino acid content, and the results are shown in Tables 1 and 2 below.

[0037] [Table 1]

[0038] [Table 2]

[0039] Although there appear to be some important differences between the different forms, three amino acids appear to be particularly important: asparagine, aspartic acid, and citrulline, which are present at significant levels relative to the total amino acid content.

[0040] According to a second aspect of the invention there is provided a method for controlling mosquito populations comprising the steps of: - placing multiple ovitraps in areas where it is desired to reduce mosquito populations; Filling the Obitrap with water and - dosing a prescribed larvicidal amount of cow urine into a given volume of water inside the ovitrap to prepare water that does not contain any separate or additional insecticide; Monitor the ovitrap and / or area to determine effectiveness A method is provided that includes:

[0041] Preferably, the targeted mosquito population is one of the subfamilies Anophelinae and Culicinae.

[0042] Culicinae is preferably Aedini, more preferably Aedes species or Culicini, more preferably Culex species.

[0043] According to a variant to the second aspect of the invention, there is provided a method for controlling mosquito populations comprising the steps of: Identifying water sources in areas where it is desired to reduce mosquito populations; - dosing a prescribed larvicidal amount of a water conditioner containing cow urine into a given volume of water that does not contain another or additional insecticide; · Monitor water and / or areas to determine effectiveness A method is provided that includes:

[0044] A source of water in an area may include any relatively small man-made object or mechanism that holds water, such as a pond, an open fish tank, or a rain gutter around a house.

[0045] Preferably, the method comprises one or more of monitoring the number of adult mosquitoes, monitoring the number of eggs laid, and / or determining the number of dead larvae.

[0046] Preferably, the method involves placing a plurality of ovitraps in an area where it is desired to reduce the mosquito population.

[0047] According to a third aspect of the present invention there is provided cow urine for use as a larvicidal agent in the population control of mosquitoes of the genus Anopheles or Culicine.

[0048] Cow urine can be used, for example, in methods to prevent the spread of diseases such as malaria and arboviral diseases, including, but not limited to, dengue fever.

[0049] Embodiments of the present invention are further described hereinafter with reference to the accompanying drawings. [Brief description of the drawings]

[0050] [Figure 1] FIG. 1 is a non-limiting flow diagram showing a process for killing larvae. [Diagram 2] 1 is a graph showing eggs laid at a first location. [Diagram 3] FIG. 1 is a graph showing OPI (Ovitrap Positivity Index) and EDI (Egg Density Index) at the first location. [Figure 4] 13 is a graph showing eggs laid at a second location. [Diagram 5] 13 is a graph showing OPI and EDI at a second location. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] Cow urine was tested in two field trials described below. Ovitrap field testing

[0052] Field trials were conducted using liquid and solid (redissolved) cow urine at two concentrations with the following treatment details: Processing Details: T1: Bioactive 1-CU (bovine urine)-10%, 15% (vol / vol) T2: Bioactive 1-Tablet (Concentrated cow urine tablet)-10%, 15% (weight / volume) Control: Water

[0053] Test location: Two different test locations were used.

[0054] Location 1 includes schools, hostels, health centres, human settlements, livestock farms and openings where mosquitoes may breed. The area was 40 acres, including aquaria containing different treatment concentrations and control water. Twenty-six ovitraps containing different treatment concentrations and control water were placed over a 3000m 2 The plants were randomly placed over a site spanning an area of ​​over

[0055] Site 2 was a 30-acre area characterized by a villa, restaurant and hotel accommodation dotted with native vegetation including shrubs, trees and large open grasslands. Twenty ovitraps containing different treatment concentrations and control water were placed over a 2000m 2 The samples were randomly placed over two locations, spanning an area of ​​over 10,000 m.

[0056] In both test locations, traps were randomly distributed by creating random numbers in each area according to a randomized complete block design (RCBD statistical design).

[0057] observation: Paper strips placed in the ovitraps for egg detection were replaced once a week, and the strips were brought into the laboratory and the number of eggs per strip was counted under a stereo binocular microscope.

[0058] Immature larvae greater than the second instar, if found in the traps, were placed in vials and used for identification to species level.

[0059] result: Location 1: The results are shown in Figure 2.

[0060] These indicate that in all treatments and in all traps, laid eggs were present from the first week of the study onwards.

[0061] In both treatments (T1 and T2), the total number of eggs and the average number of eggs were 2-3 times higher compared to the control. Both the total number and the average number of eggs per trap increased over time in the treatments and were the lowest in the control. The total number and the average number of eggs in the control traps were the lowest at week 11. At all times, the average number of eggs laid in the control traps ranged between 200 and 450. The average number of eggs was similarly high in T2 at both the 10% and 15% concentrations, exceeding 600 eggs. In T2, the number of eggs laid was the highest (over 600 eggs), even at week 11 of the field trial. Both treatments were more attractive to ovipositing female mosquitoes compared to the control traps throughout the trial. The total number and the average number of eggs laid per trap followed a similar increasing trend in both concentrations, especially in the T2 treatment at week 11. Aedes aegypti and Aedes albopictus mosquitoes were reported in all traps from week 1. Armigera species were attracted for oviposition from week 3 onwards. Culex quinquefasciatus was also attracted for oviposition from week 7 onwards.

[0062] At week 11, the contents of all traps were replaced with fresh solution for all treatments, including the control. By week 11, spawning females of Aedes albopictus and Armigera species dominated the traps, including the control. The populations of Aedes aegypti reported in the test traps due to spawning were significantly reduced by week 11. The number of adults representative of the population also showed reduced numbers of Aedes aegypti and Culex species compared to Aedes albopictus and Armigera species. Adult populations were significantly reduced in areas up to 5 acres, as was evident by sampling adults in the evening using insect nets.

[0063] Referring to Figure 3, all traps, including the control, recorded oviposition by mosquitoes from week 1 onwards. The OPI (Ovitrap Positive Index) was 100 for the treatments and above 50 for the control traps throughout the study. The EDI (Egg Density Index) increased over time with considerable variation. The EDI showed a linear step-up (trend line) indicating a positive correlation with ovitrap egg density over time. From week 2 onwards, the EDI was higher in all treatments compared to the control and the trend continued. For the 11 week data, the highest EDI was obtained for T1 (200) followed by T2 (above 150). The EDI for the control traps was always low, fluctuating between 25 and 80 throughout the study until week 9. By week 11, the EDI dropped significantly, even though all treatments and controls had an OPI above 50. At week 11, T2, C2 recorded the highest EDI of approximately 120, followed by T2, C1, which was the lowest in the control traps. Clearly, there is a significant positive correlation between EDI and time.

[0064] Location 2: With reference to Figure 4, oviposition, mostly by Aedes aegypti and Aedes albopictus, was observed in the control as well as in the treatments (T1, T2) at all concentrations. In both treatments, the total number of eggs and the average number of eggs were 2-3 times higher compared to the control. The number of eggs (average and total) increased over time in both treatments until week 10, ranging from 600 to 1400 eggs, and was the lowest in the control (less than 200 eggs). The total and average number of eggs in the control traps were the lowest in all 10 weeks of observations. At all times, the average number of eggs laid in the control traps ranged between 30 and 300. Aedes aegypti and Aedes albopictus mosquitoes were reported in all traps from week 1. Armigera species were attracted for oviposition from week 3 onwards. From week 7 onwards, Culex quinquefasciatus was also attracted for oviposition. At week 11 of the study, T1 and T2 showed increased attractiveness to oviparous females as indicated by egg counts. At week 11, the average number of eggs was over 1400 in T1 and approximately 500 in T2. ​​The average number in the control traps was 100 at week 11 of the study. Both treatments were more attractive to oviparous female mosquitoes compared to the control traps throughout the study. Adult populations were significantly reduced in areas up to 5 acres as was evident by evening sampling of adults using a net. Mosquito populations in the area around the traps were reduced and this was due to the presence of the traps. The presence of conditioned water (T1, T2) in the traps was highly attractive to prolific oviparous females of various genera and species. The presence of dogs is also favorable as it provides mosquitoes with a constant host to blood feed on. Despite this, in an area of ​​approximately 5 acres covered by grass and trees, no mosquito activity was observed even during peak evening hours (4:00 pm - 7:30 pm), undoubtedly due to population reductions resulting from the placement of ovitraps containing water conditioners.

[0065] Referring to FIG. 5, OPI (Ovitrap Positive Index) was 100 in the treatments and over 50 in the control traps throughout the study period. EDI (Egg Density Index) increased over time and showed a linear step up (trend line) indicating a positive correlation with egg density in ovitraps over time. From week 2 onwards, EDI was higher in all treatments compared to the control and the trend continued. At week 11, EDI was highest in T1 (over 300), followed by T2 (over 125) and lowest in the control (25). There is a significant positive correlation between EDI and time. Aedes aegypti and Aedes albopictus mosquitoes were reported from week 1 in all traps. Armigera species were attracted for ovitrapping from week 3 onwards. Culex quinquefasciatus was also attracted for ovitrapping from week 7 onwards. At week 11, Armigera and Aedes albopictus were more frequently reported in the traps, while occurrences of Aedes aegypti were rather occasional. A similar pattern was evident in adult samples.

[0066] Mosquito genera and species lineages reported by common ovitraps: Field trials have demonstrated that the use of cow urine is effective in attracting and killing a range of different species.

[0067] This range is illustrated below in Tables 3-1 through 3-9, which show the weekly occurrence of various genera and species of mosquitoes reported to lay eggs in ovitraps at the test site.

[0068] [Table 3-1]

[0069] [Table 3-2]

[0070] [Table 3-3]

[0071] [Table 3-4]

[0072] [Table 3-5]

[0073] [Table 3-6]

[0074] [Table 3-7]

[0075] [Table 3-8]

[0076] [Table 3-9]

[0077] Interestingly, field trials conducted at both field locations revealed a sequence in the genera and species of mosquitoes reported in the ovitraps from weeks 1 to 8. Indeed, the patterns were highly consistent across locations, suggesting that the traps became increasingly attractive to oviparous females of a diverse group of mosquitoes, continuing to capture significant numbers of eggs laid 8 weeks after water conditioning.

[0078] The first species of mosquitoes attracted to the traps at both test locations, present from week 1, were Aedes albopictus and Aedes aegypti. These continued to be reported until week 9. From week 3 onwards, the traps also attracted a new genus of mosquitoes, namely, Armigera species. Another significant fact emerging from our study is that the traps attracted Culex quinquefasciatus mosquitoes from week 7 of the field trial initiation, which was true for both locations. quinquefasciatus is a vector of lymphatic filariasis and arboviruses, including St. Louis encephalitis virus and West Nile virus. Anophonles spp. were also detected.

[0079] Conclusion: Both CU and tablets were highly effective in attracting ovipositing female mosquitoes for egg laying. The attraction was evident from the higher oviposition rate in them compared to the control traps during the trial period. The traps attracted ovipositing females of Aedes aegypti, Aedes albopictus, Armigera species, Culex quinquefasciatus and Anophonles species as evident from the identification of larvae collected from the traps. Feedback from people living at both trial sites also suggests that mosquito activity was reduced in open areas. An important feature is that both treatments were favorable for the prevention of egg laying even at the 10th week of the trial. The effect of the treatments that differentiated them from water was not negligible and this effect persisted even at the 10th week of the trial. In both locations, in an area of ​​about 5 acres covered by grass and trees, the applicant found no mosquito activity even during the evening peak hours (4:00 PM - 7:30 PM), which was undoubtedly due to the reduction in population due to the placement of ovitraps containing water conditioners. The attractiveness of the conditioned water remained effective in L2 but decreased in L1. The fact that the population density index (EDI, adult abundance) was always lower in L1 compared to L2 cannot be ignored. This study clearly shows that the cow urine and cow urine tablets used here for water conditioning remain attractive / effective even beyond 10 weeks, which is very significant.

[0080] Based on this finding, the present method suggests refilling the ovitrap with conditioned water every 8-12 weeks, for example, every other month or four times a year.

[0081] In sum, this experiment shows that cow urine placed in multiple ovitraps per acre effectively reduced populations in less than 10 weeks by attracting adults to lay eggs at high densities, disrupting the disease vector life cycle and resulting in a net reduction in larvae and adults.

Claims

1. An ovitrap comprising a container which is filled with water in use, an oviposition surface on which mosquitoes settle and lay eggs in said water, characterized in that the ovitrap contains a water conditioner which is also larvicidal such that in use the trap does not contain any added insecticide.

2. 10. The ovitrap of claim 1, comprising cow urine as a water conditioner and larvicidal agent.

3. 3. The ovitrap of claim 2, wherein the cow urine is from Bos indicus or Zebu.

4. 4. The ovitrap of claim 2 or 3, comprising significant levels of phenols, flavonoids and amino acids including asparagine, aspartic acid and citrulline relative to the total amino acid content.

5. 4. The ovitrap of claim 2 or 3, which comprises, besides water, 40-60% by weight of urea, and 40-60% by weight of other components including minerals, salts, hormones and enzymes.

6. 5. The ovitrap of claim 3 or 4, wherein the water conditioner and larvicidal agent are in unit dosage form.

7. 7. The ovitrap of claim 6, wherein the water conditioner and larvicidal agent are in tablet or liquid form with a metered dispenser.

8. 1. A method for controlling mosquito populations comprising: placing a plurality of ovitraps in an area where it is desired to reduce the mosquito population; filling the ovitrap with water; dispensing a prescribed larvicidal amount of a water conditioner comprising cow urine into a volume of water within said ovitrap to condition said water free of additional or different insecticides; monitoring said ovitraps and / or regions to determine efficacy; and

9. 1. A method for controlling mosquito populations comprising: identifying a source of water in an area where it is desired to reduce the mosquito population; administering a prescribed larvicidal amount of cow urine to a given volume of water that does not contain any separate or additional insecticides; monitoring said water and / or area to determine efficacy; and

10. 10. The method of claim 8 or 9, wherein the cow urine is from Bos indicus or Zebu.

11. 10. The method of claim 8 or 9, wherein the targeted mosquito population is from the subfamily Anophelinae.

12. 10. The method of claim 8 or 9, wherein the targeted mosquito population is from the subfamily Culicinae.

13. 12. The method of claim 11, wherein the targeted mosquito population is from the genus Anopheles and is for protection against malaria in humans.

14. 13. The method of claim 12, wherein the targeted mosquito population is from the genus Culicine and is for protection against yellow fever or dengue fever.

15. 15. The method of any one of claims 8 to 14, wherein said monitoring comprises one or more of monitoring the number of adult mosquitoes, monitoring the number of eggs laid, and / or determining the number of dead larvae.

16. 16. The method of any one of claims 8 to 15, comprising placing multiple ovitraps per acre.

17. Cow urine for use as a larvicidal agent in the population control of mosquitoes of the genus Anopheles or Culicine.

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