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.

Local Copepod Collections:

One hundred thirty adult female M. viridis 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 M. viridis (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. Paramecium caudatum were provided ad libitum 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 Ae. albopictus Colony Care:

A colony of Ae. albopictus 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). Ae. albopictus 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 M. viridis were collected from the field, Ae. albopictus 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 ad libitum. 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 M. viridis 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 Ae. albopictus 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 Ae. albopictus 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 M. viridis 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 M. viridis 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).

References

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. Plos One, 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. Environmental Research, 91, 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. International Journal of Biometeorology, 51, 525-540.

SIEGEL, J. P., NOVAK, R. J. & RUESINK, W. G. 1994. Relationship between wing length and dry-weight of mosquitoes. Journal of the American Mosquito Control Association, 10, 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. Journal of the American Mosquito Control Association, 8, 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. Journal of Vector Ecology, 21, 186-191.

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