DOI: 10.5061/dryad.vmcvdncv7
title: No impact of biocontrol agent’s predation cues on development time or size of surviving Aedes albopictus under optimal nutritional availability

description: Cyclopoid copepods have been applied successfully to limit populations of
 highly invasive Aedes albopictus mosquitoes that can transmit diseases of
 public health importance. However, there is concern that changes in
 certain mosquito traits, induced by exposure to copepod predation, might
 increase the risk of disease transmission. In this study, third instar Ae.
 albopictus larvae, hereafter referred to as “focal individuals”, were
 exposed to Megacyclops viridis predator cues associated with both
 consumption of newly-hatched mosquito larvae and attacks on focal
 individuals. The number of newly-hatched larvae surrounding each focal
 larva was held constant to control for density effects on size, and the
 focal individual’s day of pupation and wing length were recorded for each
 replicate. Exposing late instar Ae. albopictus to predation decreased
 their chances of surviving to adulthood, and three focal larvae that died
 in the predator treatment showed signs of melanization, indicative of
 wounding. Among surviving focal Ae. albopictus, no significant difference
 in either pupation day or wing length was observed due to copepod
 predation. The absence of a significant sublethal impact from M. viridis
 copepod predation on surviving later-stage larvae in this analysis
 supports the use of M. viridis as a biocontrol agent against Ae.
 albopictus.
description: Local Copepod Collections: One hundred thirty
 adult female <em>M. viridis</em> copepods were collected
 during the third week of September in 2019 from the edge of Longside Lake
 in Egham, Surrey, UK (N 51° 24.298’, W 00° 32.599’). Copepods were
 identified as <em>M. viridis</em> (Jurine, 1820) by
 morphology. The copepods were kept in ten 1 L containers, each holding
 approximately 500 mL of spring water (Highland Spring, UK) at a 12:12
 light/dark cycle, and 20 ± 1°C. <em>Paramecium caudatum</em>
 were provided <em>ad libitum</em> as food for the copepods,
 and boiled wheat seeds were added to the containers to provide a food
 source for the ciliates (Suarez et al., 1992).
 Temperate <em>Ae. albopictus</em> Colony
 Care: A colony of <em>Ae. albopictus</em>
 mosquitoes (original collection Montpellier, France 2016 obtained through
 Infravec2) was maintained at 27 ± 1°C, 70% relative humidity, and a 12:12
 light/dark cycle. Larvae were fed fish food (Cichlid Gold Hikari®, Japan),
 and adults were given 10% sucrose solution and horse blood administered
 through a membrane feeding system (Hemotek®, Blackburn, UK). <em>Ae.
 albopictus </em>eggs were collected from the colony on filter papers
 and stored in plastic bags containing damp paper towels to maintain
 humidity. Experimental Procedure: On
 the morning of the tenth day after the last <em>M.
 viridis</em> were collected from the field, <em>Ae. albopictus
 </em>larvae were hatched over a 3-hour period at 27 ± 1°C. The
 hatching temperature was kept high, relative to the temperature of the
 experiment (20 ± 1°C), to maximize the yield of larvae over a
 semi-synchronous period. Stored egg papers were submerged in 3 mg/L
 nutrient broth solution (Sigma-Aldrich © 70122 Nutrient Broth No 1) and
 oxygen was displaced by vacuum suction for 30 min. Immediately following
 oxygen displacement, ground fish food (Cichlid Gold Hikari®, Japan) was
 added <em>ad libitum</em>. After three hours, 500 larvae were
 counted and placed into spring water to dilute residual food from the
 hatching media. These focal larvae were then placed in a 1 L container
 with 600 mL of spring water and twelve pellets (each 50 mg) of fish food
 at 20 ± 1°C. The water and fish food were changed every other
 day. The focal larvae were held at a constant
 temperature of 20°C because a previous median regression showed that
 summer temperatures in South East England rose from 14.9°C to 17.0°C
 between 1971 and 1997 (Donaldson et al., 2003). This warming trend is very
 likely to continue, with the London climate projected to resemble that of
 present-day Barcelona by 2050 (Bastin et al., 2019). The average summer
 maximum temperature for the Greater London area from 1976 to 2003 was
 22.3°C (Gosling et al., 2007); therefore, 20°C is within the range of
 realistic summer temperatures to be experienced in South East England
 during the next few decades.  On the sixth day of focal
 larvae development, 70 female <em>M. viridis</em> copepods
 were each placed in a Petri dish (diameter: 50 mm, height: 20.3 mm)
 holding 20 mL of spring water to begin a 24-hour starvation period. A
 second set of <em>Ae. albopictus</em> eggs were hatched
 according to the previously described method, except that the hatch was
 held at 27 ± 1°C for 18 hours. On the seventh day of
 focal larvae development, 140 third instar focal <em>Ae.
 albopictus</em> larvae were each placed in a Petri dish holding 20
 mL of spring water, a 50 mg pellet of fish food, and four first instar
 larvae from the hatch that was started on the previous day. A subset of 30
 third instar focal larvae were preserved in 80% ethanol for head capsule
 width measurements (Teng and Apperson, 1996). Seventy starved <em>M.
 viridis</em> copepods were then introduced to 70 out of the 140
 Petri dishes. Each day, the number of surviving first
 or second instar larvae in each of the 140 Petri dishes was recorded,
 surviving first or second instars were removed, and four new first instars
 from the 18-hour hatch started on the previous day were added to each
 replicate. The status (dead or alive) of each focal larva was recorded
 daily, and in predator treatment replicates, the status of the copepod was
 also recorded. In the case of a focal larva death, the larva was preserved
 in 80% ethanol, and that replicate was removed from further observation.
 In the case of a copepod death, the copepod was preserved in 80% ethanol,
 and a new adult female <em>M. viridis</em> copepod from the
 September field collections was randomly chosen to replace it.
 Pupation among focal individuals was recorded each day at 18:00
 hrs. Pupae were transferred to 10 mL of spring water in a graduated
 cylinder with a mesh cover for emergence. Emerged adults were frozen at
 -20°C. Wings were removed and measured as a proxy for body size (Siegel et
 al., 1994). <strong>References</strong> BASTIN, J. F., CLARK, E., ELLIOTT, T., HART, S., VAN DEN HOOGEN, J., HORDIJK, I., MA, H. Z., MAJUMDER, S., MANOLI, G., MASCHLER, J., MO, L. D., ROUTH, D., YU, K. L., ZOHNER, C. M. & CROWTHER, T. W. 2019. Understanding climate change from a global analysis of city analogues. <em>Plos One,</em> 14. DONALDSON, G. C., KEATINGE, W. R. & NAYHA, S. 2003. Changes in summer temperature and heat-related mortality since 1971 in North Carolina, South Finland, and Southeast England. <em>Environmental Research,</em> 91<strong>,</strong> 1-7. GOSLING, S. N., MCGREGOR, G. R. & PALDY, A. 2007. Climate change and heat-related mortality in six cities Part 1: model construction and validation. <em>International Journal of Biometeorology,</em> 51<strong>,</strong> 525-540. SIEGEL, J. P., NOVAK, R. J. & RUESINK, W. G. 1994. Relationship between wing length and dry-weight of mosquitoes. <em>Journal of the American Mosquito Control Association,</em> 10<strong>,</strong> 186-196. SUAREZ, M. F., MARTEN, G. G. & CLARK, G. G. 1992. A simple method for cultivating freshwater copepods used in biological control of Aedes aegypti. <em>Journal of the American Mosquito Control Association,</em> 8<strong>,</strong> 409-412. TENG, H. J. & APPERSON, C. S. 1996. Identification of larval instars of Aedes albopictus (Skuse) and Aedes triseriatus (Say) (Diptera: culicidae) based on head capsule size. <em>Journal of Vector Ecology,</em> 21<strong>,</strong> 186-191.
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