EFFECT OF LARVAL NUTRITION AND REARING TEMPERATURE ON AEDES AEGYPTI (DIPTERA: CULICIDAE) ADULT PHYSIOLOGY MEREDITH G. MORROW

INTRODUCTION

Larger mosquito body size has been associated with increased longevity, biting persistence, fecundity, and vector capability, and is therefore an important measure of fitness (Nasci, 1986; Klowden et al.., 1988; Landry et al.., 1988; Nasci, 1991; Siegel et al.., 1992). The conditions under which larval mosquitoes are maintained can profoundly affect the physiology of the resulting adults. Using Aedes aegypti as a model, I determined how adult mosquito physiology is affected by larval nutrition and environmental temperature by using the standard assessment of wing length, L (Siegel et al.., 1992).

MATERIALS AND METHODS Houston strain Aedes aegypti eggs were obtained from the USUHS insectary and were vacuum hatched for approximately 15 minutes. Fifty larvae were pipetted into twenty-seven cups, each filled with an equal amount of tap water. A set of three cups were placed in an environment with an average temperature of 17.6ºC, 27.8ºC, and 32.7ºC. While the 27.8ºC rearing temperature had a controlled photoperiod of 12:12 (L:D), the 32.7ºC temperature lacked light entirely and the photoperiod for the 17.6ºC environment was approximately 10:14 (L:D) on weekdays and approximately 7:17 (L:D) on weekends. Water temperature, larval development, and larval mortality were recorded daily.

To test the effect of varying nutrition levels on growth, a subset (three cups) of larvae in each temperature were underfed, optimally fed, and overfed a mixture of ground fish food. Underfed larvae received 0.05mg per day for first and second instars and 0.1mg per day for third and fourth instars. Optimally fed larvae received 0.2mg per day for first and second instars, while third and fourth instars received 0.5mg per day (TunLin et al.., 2000). Overfed larvae received 0.4mg per day for first and second instars, and 1.0mg per day for third and fourth instars.

To determine the effects of nutrition and temperature on adult growth, wing lengths of adult mosquitoes were measured and mean ± SE wing length (mm) were calculated (Nasci, 1991; Landry et al.., 1988). Twelve females and twelve males (216 total) were sampled from each temperature and diet type. One wing was chosen randomly and was removed from the mosquito and placed on a slide. The wing was measured from the distal end of the axial inclusion to the apical margin, not including the fringe (Van Den Heuvel, 1963). Wings were measured using a dissecting microscope and an ocular ruler. Mosquitoes with both wings worn were not measured.

RESULTS 17.6ºC Environment: Larval development differed in each of the three separate environments and nutrition states. While the 17.6ºC environment produced larger larvae in the overfed and optimally fed groups, there was also a higher rate of mortality in each of the three subsets of nutrition levels at this temperature compared to the two other environments. Larval

2

development occurred more quickly in the overfed and optimally fed subsets with the first pupae emerging from both groups 12 days after hatching. Pupae from the underfed group first emerged 15 days after hatching.

The range of wing lengths for females in the underfed group was calculated to be 3.00-3.90mm with a mean wing length of 3.46mm, and those for males ranged from 2.302.80mm with a mean wing length of 2.5mm. Wing lengths for females in the optimally fed subset varied from 3.00-3.70mm with a mean wing length of 3.50mm, and males varied from 2.10-3.00mm with a mean wing length of 2.58mm. Wing lengths of the overfed females ranged from 2.60-3.70mm with a mean wing length of 3.25mm, while males ranged from 2.00-2.70mm with a mean wing length of 2.38mm.

32.7ºC Environment: Similar to the 17.6ºC environment, overfed and optimally fed larvae in the 32.7ºC environment were larger, however, unlike the 17.6ºC larvae, they experienced less mortality compared to the underfed larvae. Pupae first emerged from the overfed subset 5 days after hatching followed by the optimally fed subset pupating the following day. The underfed subgroup first pupated 8 days after hatching.

The range of wing lengths for females in the underfed group was calculated to be 2.40-2.70mm with a mean wing length of 2.56mm, and those for males ranged from 1.602.50mm with a mean wing length of 2.00mm. Wing lengths for females in the optimally fed subset varied from 2.60-3.10mm with a mean wing length of 2.88mm, and males varied from 1.80-2.20mm with a mean wing length of 2.04mm. Wing lengths of the

3

overfed females ranged from 2.00–3.30mm with a mean wing length of 2.82mm, while males ranged from 1.70–2.40mm with a mean wing length of 2.08mm.

27.8ºC Environment: Unlike the two other temperature groups, the 27.8ºC environment had a similar range of mortality in all subsets. However, the optimally fed group had a quicker development time and first pupated 6 days after hatching, followed by the overfed and underfed at 7 and 8 days respectively.

The range of wing lengths for females in the underfed group was calculated to be 2.50-3.00mm with a mean wing length of 2.78mm, and those for males ranged from 1.902.60mm with a mean wing length of 2.24mm. Wing lengths for females in the optimally fed subset varied from 3.00-3.50mm with a mean wing length of 3.21mm, and males varied from 2.00-2.30mm with a mean wing length of 2.10mm. Wing lengths of the overfed females ranged from 3.20–3.50mm with a mean wing length of 3.34mm, while males ranged from 2.00–2.40mm with a mean wing length of 2.18mm.

The largest female wing lengths in this study were from the 17.6ºC, optimum fed female group of mosquitoes (mean±SE = 2.58±0.07), followed by underfed females at the same temperature (3.46±0.09). Parallel to the female measurements, the largest male wing lengths were from the 17.6ºC, optimum fed subset (2.58±0.07), followed by underfed males at the same temperature (2.50±0.04). Wing length in both sexes steadily declined as temperature increased.

4

The underfed groups of both males and females of the highest rearing temperature (32.7ºC) produced the smallest adult mosquitoes. Wing length also steadily increased as the diet increased to optimally fed and overfed in both males and females at this temperature. However, there is a discrepancy as to whether or not optimum fed versus overfed larvae produce larger adults at the 27.8ºC rearing temperature. While females followed the trend of increasing in size as the amount of food offered increased, the males did not follow this pattern. Instead, the underfed males at this temperature produced the largest wing lengths, followed by overfed males.

Table 1. Wing lengths of male and female Aedes aegypti (n=12) Temperature/Diet Mean±SE wing length (mm) Range (mm) _______________________________________________________________________________________________ 17.6ºC Underfed Males 2.50±0.04 2.30-2.80 Females 3.46±0.09 3.00-3.90 Optimum Males 2.58±0.07 2.10-3.00 Females 3.50±0.07 3.00-3.70 Overfed Males 2.38±0.08 2.00-2.70 Females 3.25±0.10 2.60-3.70 27.8ºC Underfed Males Females Optimum Males Females Overfed Males Females 32.7ºC Underfed Males Females Optimum Males Females Overfed Males Females

2.24±0.07 2.78±0.05

1.90-2.60 2.50-3.00

2.10±0.03 3.21±0.05

2.00-2.30 3.00-3.50

2.18±0.04 3.34±0.03

2.00–2.40 3.20–3.50

2.00±0.07 2.56±0.03

1.60-2.50 2.40-2.70

2.04±0.04 2.88±0.06

1.80-2.20 2.60-3.10

2.08±0.06 2.82±0.10

1.70–2.40 2.00–3.30

5

DISCUSSION

Maintaining proper nutrition and optimum environmental temperature throughout larval development will have positive effects for the entirety of the mosquito life. Optimizing nutrition, photoperiod, competition, and temperature will result in healthier larvae and a more productive colony (Nasci, 1991).

This study found that both rearing temperature and larval diet has a definitive effect on adult physiology. Larger mosquitoes were found in the cooler temperature range (17.6ºC), while smaller wing length measurements were observed as rearing temperature increased. The larvae in the 32.6ºC temperature also developed much more quickly compared to the other environments, while mosquito subsets at 17.6ºC took the longest to pupate perhaps growing larger due to the increased feeding time. The 27.8ºC group had less mortality in all subgroups, while each subgroup also pupated one day apart. At the highest rearing temperature, it was found that diet had a more directly, observable effect on size of males, while diet had a more directly, observable effect on females at 27.8ºC. The inconsistency between male and female wing lengths in the higher rearing temperatures may be due to differences in photoperiods between the two temperatures as photoperiod was not comparable between the three temperature groups. Controlling the photoperiods in each group and increasing the sample size of adult wing length (n>12) in each temperature would increase the power, reliability and precision of wing length measurements if performed again in the future.

6

While the quality of diet was not considered in this study, quantity of larval food was found to be of significant importance in both adult size and larval mortality rates at higher rearing temperatures. Overfeeding larvae will often lead to high larval mortality, as was witnessed in the highest rearing temperature (Lillie and Nakasone, 1982; Reisen, 1975). Conversely, underfed larvae will result in the production of smaller adults which may have implications in how much blood they may be able to ingest, thus effecting fecundity and vector capability as adults (Arrivillaga & Barrera, 2004; Briegel, 1990; Landry et al.., 1988). Therefore, it would be reasonable to assume that underfed mosquitoes reared at higher temperatures may not make effective vectors due to their small size as was observed in this study. Nasci (1991) showed that large adult Aedes aegypti from larvae fed on optimal diets were more likely to engage in host-seeking behavior, and were more persistent biters than were small females that were underfed. Telang et al.. (2005) also found that optimally fed larvae were larger and, after ingesting a blood meal, matured more eggs compared with blood-fed females of low-fed larvae. In addition, the longer life span of large females increases the probability that these females will contact and successfully feed upon a host (Briegel, 1990; Nasci, 1991). For that reason, the optimally fed larvae in this study which were reared at the lowest temperature may have produced more-likely competent vectors (for the reasons stated above) since this combination produced the largest adults. ACKNOWLEDGMENTS

I would like to thank Nicole L. Achee and John P. Grieco for supplying all that was necessary to rear the mosquitoes for this experiment, and David M. Claborn for supplying the ocular ruler used to measure the mosquito wing lengths.

7

LITERATURE CITED Arrivillaga J, Barrera R (2004) Food as a limiting factor for Aedes aegypti in water-storage containers. Journal of Vector Ecology. 29: 11-20. Briegel H. (1990) Fecundity, Metabolism, and Body Size in Anopheles (Diptera: Culicidae), Vectors of Malaria. Journal of Medical Entomology. 27: 839-850. Klowden et al.. (1988) Effects of Larval Nutrition on the Host-Seeking Behavior of adult Aedes aegypti mosquitoes. Journal of the American Mosquito Control Association. 4(1): 73-75. Landry et al.. (1988) Adult body size and survivorship in a field population of Aedes triseriatus. Journal of the American Mosquito Control Association. 4(2): 121-128. Lillie TH, Nakasone RI. (1982) An evaluation of commercial diets for rearing Wyeomyia smithii. Mosquito News. 42: 225-231. Nasci RS. (1991) Influence of Larval and Adult Nutrition on Biting Persistence in Aedes aegypti (Diptera: Culicidae). Journal of Medical Entomology. 28(4): 522-526. Nasci RS. (1986) The Size of Emerging and Host-Seeking Aedes aegypti and the Relation of Size to Blood-feeding success in the field. Journal of the American Mosquito Control Association. 2(1): 61-62. Reisen WK. (1975) Intraspecific competition in Anopheles stephensi Liston. Mosquito News. 35: 473-482.

8

Siegel et al.. (1992) Statistical Appraisal of the Weight-Wing Length Relationship of Mosquitoes. Journal of Medical Entomology. 29(4): 711-714. Telang et al.. (2005) Effects of larval nutrition on the endocrinology of mosquito egg development. The Journal of Experimental Biology. 209: 645-655. Tun-Lin et al.. (2000) Effects of temperature and larval diet on development rates and survival of the dengue vector Aedes aegypti in north Queensland, Australia. Medical and Veterinary Entomology. 14(1): 31-37. Van Den Heuvel, M.J. (1963) The effect of rearing temperature on the wing length, thorax length, leg length and ovariole number of the adult mosquito, Aedes aegypti (L.). Trans. R. Entomological Society Lond. 115: 197-216.

9