Electronic supplementary material for Satyrization without evidence

Electronic supplementary material for
Satyrization without evidence of successful insemination from interspecific mating between
invasive mosquitoes
María C. Carrasquilla* and L. Philip Lounibos
Florida Medical Entomology Laboratory, University of Florida
*author for correspondence; email: [email protected]
Absence of heterospecific sperm in spermathecae and refractoriness to mating:
-Brief explanation of the technique that was used for detection of insemination status in live females:
Using a dissecting microscope, each female lightly anesthetized with chloroform, was placed ventral
surface up on a glass slide. The tip of the abdomen of the female was located close to a drop of water.
Presence or absence of sperm in the spermathecae was visualized by phase contrast microscopy
through the female’s distended abdomen, after the intersegmental membranes were stretched by ventral
placement of a glass cover slip. After sperm check, the cover slip was removed and each female was
placed in a small container. If the abdomen failed to distend properly and the spermathecae were not
observable, the female was discarded. Females usually recovered from the procedure in 30–60 min.
Each female that was negative for sperm was exposed to two virgin A. aegypti males for 24 hours. After
exposures with conspecific males, healthy females were dissected to check for presence or absence of
sperm in spermathecae. A female was considered healthy if she was able to stand upright and fly [1].
- Sham-treated control: The same procedures explained above, that were used to detect insemination
status in live females in the interspecific-conspecific treatment, were applied to virgin females.
Subsequently females were housed with conspecific males, and dissected.
Transfer of semen:
-Stable isotope labeling:
Mosquitoes were labeled with 15N-glycine (NLM-202-1; Cambridge Isotope Laboratories, Inc.), following
procedures described by Helinski et al. [2], with some modifications. To obtain males labeled with 15N,
eggs were hatched in a vacuum, and 200 first instar larvae were placed in a tray with 1 liter of Milli-Q
water. Larvae received 68 mg of
on day 1 and 20 mg on day 3 (25% enrichment based on
nitrogen content in yeast and lactalbumin). Larvae also received a standard diet (a 1:1 mixture of
lactalbumin and yeast) according to the following schedule: day one: 38 mg, day three: 75 mg, day four:
113 mg, day five: 150 mg, day six: 113 mg. To obtain unlabeled females, larvae were reared with
standard diet (a 1:1 mixture of lactalbumin and yeast).
-Stable isotope analysis: Isotope detection was performed by the Light Stable Isotope Mass Spectrometry
Lab in the Department of Geological Sciences at the University of Florida. Female mosquitoes were
loaded into tin capsules and combusted in a Carlo Erba NA 1500 CNS elemental analyzer to separate N2
from CO2. The sample gas next passed into a ConFlo II preparation system and into the inlet of a Thermo
Electron Delta V Advantage isotope ratio mass spectrometer where the sample gas was measured
relative to laboratory reference N2 and CO2 gases. Nitrogen isotopic results are expressed in standard
delta notation relative to Air [3].
-Data analysis: A female was considered labeled when her delta value was above a conservative
threshold [2-6]. This threshold was defined as three standard deviations above the mean of the reference
value, based on measurements of unlabeled females. Each of the replicates with its controls was
performed at a different time, with separate threshold values established and applied for each replicate.
Supplementary references
1. Carrasquilla MC, Lounibos LP. 2015 Detection of insemination status in live Aedes aegypti females. J.
Insect. Physiol. 75, 1-4. (doi.org/10.1016/j.jinsphys.2015.01.015)
2. Helinski M, Valerio L, Facchinelli L, Scott TW, Ramsey J, Harrington L. 2012 Evidence of polyandry in
semifield exposures. Am. J. Trop. Med. Hyg. 86: 635-641. (doi.org/10.4269/ajtmh.2012.11-0225)
3. Hübschmann HJ. 2009 Handbook of GC/MS: fundamentals and applications, 2nd edition, Germany:
Wiley-VCH Verlag GmbH & Co. KGaA.
4. Helinski MEH, Hood-Nowotny R, Mayr L, Knols BGJ. 2007 Stable isotope-mass spectrometric
determination of semen transfer in malaria mosquitoes. J. Exp. Biol. 210, 1266-1274.
5. Helinski MEH, Hood RC, Gludovacz D, Mayr L, Knols BGJ. 2008 A
stable isotope semen label to
detect mating in the malaria mosquito Anopheles arabiensis Patton. Parasites & Vectors 1, 19.
6. Macneale KH, Peckarsky BL, Likens GE. 2005 Stable isotopes identify dispersal patterns of stonefly
populations living along stream corridors. Freshwater Biology 50, 1117–1130.
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