“Singing in the Tube”—audiovisual assay of plant oil repellent activity against mosquitoes (Culex pipiens)

Abstract

Plant essential oils have been suggested as a promising alternative to the established mosquito repellent DEET (N,N-diethyl-meta-toluamide). Searching for an assay with generally available equipment, we designed a new audiovisual assay of repellent activity against mosquitoes “Singing in the Tube,” testing single mosquitoes in Drosophila cultivation tubes. Statistics with regression analysis should compensate for limitations of simple hardware. The assay was established with female Culex pipiens mosquitoes in 60 experiments, 120-h audio recording, and 2580 estimations of the distance between mosquito sitting position and the chemical. Correlations between parameters of sitting position, flight activity pattern, and flight tone spectrum were analyzed. Regression analysis of psycho-acoustic data of audio files (dB[A]) used a squared and modified sinus function determining wing beat frequency WBF ± SD (357 ± 47 Hz). Application of logistic regression defined the repelling velocity constant. The repelling velocity constant showed a decreasing order of efficiency of plant essential oils: rosemary (Rosmarinus officinalis), eucalyptus (Eucalyptus globulus), lavender (Lavandula angustifolia), citronella (Cymbopogon nardus), tea tree (Melaleuca alternifolia), clove (Syzygium aromaticum), lemon (Citrus limon), patchouli (Pogostemon cablin), DEET, cedar wood (Cedrus atlantica). In conclusion, we suggest (1) disease vector control (e.g., impregnation of bed nets) by eight plant essential oils with repelling velocity superior to DEET, (2) simple mosquito repellency testing in Drosophila cultivation tubes, (3) automated approaches and room surveillance by generally available audio equipment (dB[A]: ISO standard 226), and (4) quantification of repellent activity by parameters of the audiovisual assay defined by correlation and regression analyses.

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Abbreviations

a :

Limit of mosquito movement determined by regression analysis with logistic curve

AT:

Length of Drosophila cultivation tube above water level determined by geometry

b :

Inverse of steepness of logistic curve

c vz :

Repelling velocity constant defined by parameters of logistic regression (c vz = 0.5*a/t 0)

D xy :

Distance between chemical and mosquito in horizontal plane

D z :

Repelling distance z-component (along vertical and longitudinal axes of tube) between chemical and mosquito, measured between net (closing tube and carrying test chemical on antibiotic assay disc) and mosquito body without proboscis and appendices

f :

Frequency in equations of sinus curves sin-1, sin-2, sin-3, sin-4, sin-5, sin-6, sin-7, sin-8, sin-9

ff:

Fundamental frequency

FTT:

Flight total time (%)

HO:

Harmonic order

IROFFI:

Initial rate of first flight induction

IROFFI:

1/TOFFI with TOFFI time of first flight induction during 120 min

n F :

Number of flights during 120 min

n F-lin:

Number of flights during 120 min resolved on linear scale

n F-log:

Number of flights during 120 min resolved on common logarithmic scale

POF:

Period of time of flight

P(f):

Significance of frequency of regressions with sin-1, sin-2, sin-3, sin-4, sin-5, sin-6, sin-7, sin-8, sin-9

P(c vz):

Significance of repelling velocity constant (c vz) by variance analysis of regression: D z (t), a/(1 + exp(−(t − t 0)/b)) with c vz = 0.5*a/t 0

r s :

Non-parametric Spearman correlation coefficient

sin-1(x):

a*sin(2πx/f + c)

sin-2(x):

y 0 + a*sin(2πx/f + c)

sin-3(x):

a*sin(2πx/f + c)2

sin-4(x):

y 0 + a*sin(2πx/f + c)2

sin-5(x):

a*e (−x/d)*sin(2πx/f + c)

sin-6(x):

y 0 + a*e (−x/d)*sin(2πx/f + c)

sin-7(x):

a*sin(π(x − x 0)/f)

sin-8(x):

a*sin(π(x − x 0)/f)2

sin-9(x):

a*e (−x/c)*sin(π(x − x 0)/f)

t 0 :

Time to push mosquito to half-maximum repelling distance z-component (0.5*a) after placing an antibiotic assay disc with repellent activity on net cover of tube

t x :

Time points of readings of D z (min ± 15 s): 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120

TOFFI:

Time of first flight induction during 120 min

V :

Volume of flight tone (dB[A]: ISO standard 226: 2003 revision)

WAV:

Waveform Audio File Format

WBF:

Wing beat frequency

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Acknowledgments

Support by OEAD, Austrian Academic Exchange Service South East Asia Programme TSA Post Doc ICM-2013-05667, to C.W. is gratefully acknowledged. Special thanks go to Peter Ogufere for help in informatics. We thank Helga Ennemoser for washing the Drosophila cultivation tubes.

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Correspondence to Wolfgang Pfeiffer.

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Adams, T.F., Wongchai, C., Chaidee, A. et al. “Singing in the Tube”—audiovisual assay of plant oil repellent activity against mosquitoes (Culex pipiens). Parasitol Res 115, 225–239 (2016). https://doi.org/10.1007/s00436-015-4739-x

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Keywords

  • Audiovisual assay of repellent activity against mosquitoes
  • Culex pipiens
  • DEET (N,N-diethyl-meta-toluamide)
  • Plant essential oils
  • Repelling velocity constant defined by logistic regression