Ecological Entomology (1994) 19, 74-78

Energy budget of swarming male mosquitoes BOAZ Y U V A L , M E R R Y L . HOLLIDAY-HANSON and R O B E R T K . California, U.S.A.

W A S H I N O Department of Entomology, University of California, Davis,

Abstract. 1. The objective of this study was to determine, in the field, the energetic costs of swarming for male Anopheles freeborni (Diptera: Culicidae). By comparing the caloric contents of resting males to marked males captured after swarming, we established when sugar feeding takes place, what energy source is used to fuel swarming flight, and how much energy is invested in this activity.

2. Sugar-feeding takes place sometime during the night after swarming is concluded. Nectar sugars are therefore not immediately available to fuel flight. Stored sugars (trehalose and glucose) and glycogen are the primary sources of energy for flight. Lipids are not used to fuel flight but may be used in resting metabolism.

3. Male size is not related to feeding success. For males of all sizes, swarming consumes more than 50% of available calories. Accordingly, the ability of an individual to find and exploit nectar sources will greatly affect reproductive success.

Key words. Anopheles, sugar-feeding, swarming, reproductive success.

Introduction

Swarming behaviour has evolved repeatedly in various groups of insects (Sullivan, 1981; Cooter, 1989; Svensson & Petersson, 1992). Typically, males predominate in swarms; females approach the swarm, acquire a mate, and promptly depart in copula. The benefits of swarming are obviously reproductive (Parker, 1978), and the deter- minants of mating success in swarms have been examined in several species (Thornhill, 1980; Flecker et al., 1988; Petersson, 1989, 19Wa, b; Neems et al., 1990; Yuval ct a / . , 1993).

Little, however, is known about the costs of swarming. Males that swarm invest energy in flight and are potentially exposed to predators. Predation on swarming chironomids (Neems ct a / , , 1992) and anophelines (Yuval & Bouskila, 1993) has recently been analysed. Few studies have con- sidered the energetic investment in swarming. Neems et a / . (1990) have shown in the laboratory that larger individuals of the midge Chironomus plumosus can sustain flight longer than small ones, and that feeding on carbohydrates enhances the ability of male chironomids to fly. Petersson ( 19YOa) demonstrated a correlation between fat reserves and mating success of caddis flies, who do not feed as

Corrcspondcncc: Dr B. Yuval. Dcpartrnent of Entomology, Hebrew University, P.O. Box 12, Rehovot 76100, Israel.

adults. However, there is no precise information on the energetic costs of swarming in the field or the relation of this investment to the energy reserves available to males.

In the central. valley of California, swarms of male Ariopheles freeborni mosquitoes assemble on most even- ings from July to late September. Swarms form 5- LOmin after sunset, last 30-35 min and disperse when twilight ends or earlier when conditions are windy. Numbers of individual mosquitoes in these swarms build up during the first 15-20min, and then gradually decline (Yuval r t ul., 1993). Adult mosquitoes emerge with low energy rcscrves. which they gradually build up by sugar-feeding. I n most species, both sexes feed repeatedly on plant sugars (mainly nectars) throughout their lives, and the carbohydrates thus acquired serve to sustain their daily activities (reviews by O'Meara, 1987; Yuval, 1992). Our objective in this study was to determine the energy budget of swarming male An.freeborni. By comparing the caloric content of resting males to marked males captured after swarming, we established what energy source is used to fuel swarming flight, how much energy is invested in this activity, and when sugar feeding takes place.

Materials and Methods

Field collections. Field collections were made on a farm.

74

Mosquito energy budget 75

adjacent to rice fields, near Pleasant Grove, Sutter Co., California, on three occasions in July and August 1992. We collected male mosquitoes from four behavioural groups: (1) afternoon resters, (2) early swarmers, (3) late swarmers, and (4) morning resters. To halt metabolic processes, after collection, all mosquitoes were immedi- ately placed on dry ice and later stored at -80°C. Resting mosquitoes were collected with a mechanical aspirator from a red box (3 X 1 X 1 m), which attracts mosquitoes seeking a resting site. These collections were made 1 h before dusk and 1 h after dawn on the following morning.

Swarming males were collected on the evening between the two resting collections. We have previously established that numbers of individuals in swarms increase during the first 20min, then gradually decline (Yuval et al., 1993). This pattern suggests that individuals remain in the swarm as long as they are capable of sustaining flight. The excep- tion to this rule is males that leave a swarm in copula and promptly (after about 15s) return. Initially, a sample of swarming males was taken with a sweep net 5-l0min after swarms formed. Immediately after this sampling event, males remaining in the same swarm were marked by blowing a cloud of fluorescent dust (Hercules Inc., Wilmington, Delaware) directly onto the mass of swarming mosquitoes. This technique marks 5-60% of the males (unpublished) without interfering with their behaviour. 20min after marking, all of the mosquitoes remaining in the marked swarm were captured with a sweep net and immediately frozen. In the laboratory, these mos- quitoes were illuminated with an ultraviolet light to identify the marked mosquitoes, which were retained as our sample of late swarmers. Unmarked mosquitoes were discarded. We assume (for the reasons mentioned above) that the marked individuals had remained flying in the swarm continuously from the time the swarm was marked until they were collected.

While mosquitoes swarmed, wind speed was measured with a hand-held anemometer. Fortuitously, wind speeds on the evenings we sampled were uniformly low, ranging from 0 to 0.1 m/s; therefore wind speed did not affect the amount of energy expended by swarming individuals.

Analytical methods. As an index of individual size, one wing was removed from each male and measured from its tip to the allular notch. As with many other mosquito species, this measure is significantly correlated to dry weight in An.freeborni (Yuval et al . , 1993).

To determine the exact amounts of lipids, sugars and glycogen i n individual mosquitoes, we adopted the pro- tocols described by Van Handel (1985) and Van Handel & Day (1990). Briefly, each individual male was crushed in 0.2ml of a 2% sodium sulphate solution, 1.Sml of ch1oroform:methanol (1:2) were added to the homogenate, and centrifuged at 1400 r e v h i n for 1Smin. After cen- trifugation the supernatant was divided in two parts, one for the lipid and the other for the sugar assay. The precipitate was kept for glycogen analysis. Lipids were determined by evaporating the first portion of the super- natant and adding 0.2 ml sulphuric acid, heating for 10min at YO-Y3"C, and adding 4.8 ml vanillin reagent (600mg of

vanillin dissolved in 100ml distilled water and diluted in 400ml of 85% phosphoric acid). After lomin, absorbance a t 525 nm was read on a spectrophotometer.

Sugars and glycogen were determined with the anthrone reagent (1.4% in 70% sulphuric acid). To determine fructose (the most common nectar sugar) concentrations, the second portion of the supernatant was evaporated to a volume of c. 0.1 ml t o which 4.91111 of cold anthrone were added. This reaction was incubated for 45min at room temperature and read at 625 nm. Subsequently, to deter- mine the quantity of all sugars present, the same sample was heated for 15min at 90-93°C and absorbance was read at 62.5 nm. The difference between the hot and cold anthrone readings was taken to represent the amount of storage sugars (trehalose and its precursor glucose) in each individual tested (Van Handel & Day, 1990). Glycogen concentrations were determined from the precipitate by adding 5 m l of anthrone reagent, heating for 15min. and measuring absorbance at 625 nm. Calibration curves were created by measuring the absorbance of serial dilutions of soybean oil, sucrose or glucose. Concentrations (in pg) of all four compounds were calculated from the slope of the calibration curves and transformed into calories by multiplying carbohydrates by 0.004 and lipids by 0.009.

To determine irreducible minimum quantities of the above compounds, a sample of twenty field-collected resting males was held in the laboratory in a cage with water but no nutrients. After these males died, they were prcwessed as above. Males with unrotated genitalia were occasionally encountered in the morning resting samples, but were considered teneral and not analysed.

Statistical analysis. From each behavioural category, twenty-four males were analysed for each sampling event (except late swarmers where only twenty, twenty-two and twelve marked individuals were recovered in each sampling event). Initially, the average concentrations of carbohydrates and lipids in each behavioural class were compared between samples by a one-way analysis of variance (ANOVA). As no significant differences were evident, we pooled the data from the three samples (Zar, 1984). The concentrations of lipids and carbohydrates between the different behavioural classes were then com- pared by one-way ANOVA.

Correlations between the various nutrients and indi- vidual wing length were determined by calculating Pearson's product moment coefficient of correlation (Zar, 1984).

Results

Time of sugar-feeding

Sugar-feeding took place sometime after swarming activity ceased and before males entered resting sites. Significant amounts of fructose were detected only in the males collected in the morning resting samples. These males contained significantly more fructose than those collected resting in the afternoon (one-way ANOVA, F = 40.1, P < 0.001), and those sampled when swarming began (F=52, P<O.001). The amount of fructose in

76 B. Yuval, M . L . Holliday-Hanson and R. K . Washino

1.50 Fuel for flight

0.00 late starved morning afternoon early

swarm *Warm

Fig. 1. Caloric reserves in male Anopheles freeborni collected while resting (in the morning or afternoon), while swarming, or starvcd to death in the laboratory. Late swarmers were sampled 20-25 min after the early swarm was sampled. Abbreviations: L, lipid; F, fructose; 0s. other sugars (glucose and trehalose); G, glycogen.

males resting in the late afternoon and swarming was negligible (Fig. 1). The average amounts of fructose in both groups of swarming males (sampled when they began to swarm or after 20min of swarming) were not significantly different from those found in males collected from resting sites in the late afternoon.

To establish the frequency of sugar-feeding in the popu- lation, we examined levels of nectar sugars of individual males (Fig. 2). Of those sampled in the morning collections 59.7% were nectar positive (i.e. with >10pg of fructose), whereas only 4.3% of swarming males (three individuals) contained noticeable amounts of fructose. The absence of nectar in males collected after swarming for 20min confirms our impression that they do not leave the swarm to sugar-feed and subsequently resume swarming. We conclude that sugar-feeding takes place sometime during the night after swarms disperse, and that nectar is not immediately available to fuel flight.

100

0 0-10 10-20 20-30 30-40 40-50 50-380

Fructose (micrograms/mosquito)

Fig. 2. Frequency distribution of nectar content in individual male Anopheles freeborni. The percentages of males from three behavioural categories arc grouped according to the amount of fructosc found in cach individual.

(a) Sugar. Storage sugar levels of males sampled swarm- ing late were significantly lower than those of males sampled swarming earlier (F = 27, P < 0.001), indicating that these sugars (probably in the form of glucose) are mobilized for flight (Fig. 3).

(b) Glycogen. Levels of glycogen in the males sampled after swarming for 20-25 min had decreased significantly from initial swarm levels (F=7.38, P=0.007) (Fig. 3). Thus, glycogen also serves as a fuel for flight.

0.28 0

3 U

c .-

- i! o’2’

.I 0.14 0 id - 0 0.07

0.00 morning afternoon early late swarm swaim

Fig. 3. Storage and consumption of sugars (glucose and trehalose) and glycogen by male Anopheles freeborni. Average calories (+SE) per mosquito in four behavioural groups.

(c) Lipids. The mean amount of lipids present in indi- vidual mosquitoes did not vary significantly between the behavioural groups. On average, males from the four behavioural groups had 0.79-0.89 calories stored as lipids (Fig. 1). Lipid levels were similar in the early and late swarming groups (F = 0.55, P = 0.5). Therefore it is evident that this substrate is not used to fuel flight. However, there is circumstantial evidence that lipids are used for resting metabolism: average calories from lipid in the group starved to death were only 0.37 (SD = 0.016) (Fig. 1).

Size-related cost of swarming

Lipid content was consistently and highly significantly correlated to size (i.e. wing length) in all groups except the late swarmers (Pearson’s r = 0.33 for morning resters, 0.38 in the afternoon and 0.44 in the early swarm sample). Storage sugar content was also correlated to size in the afternoon resters and early swarmers, but the significance levels of these values were barely under the 0.05 threshold. Glycogen and nectar concentrations were never found to correlate to size.

To determine the size-related cost of swarming, we divided the mosquitoes from the four behavioural groups

Mosquito energy budget 77

Table 1. Average fuel reserves (calories) (n: SE) of resting and swarming male Anopheles freeborni in ascending size classes.

Size class*

Time sampled 1 I1 111 IV

Morning 1.08 (7; 0.15) 0.55 (24; 0.07) 0.73 (26: 0.07) 0.62 (13; 0.12) Afternoon 0.66 (5; 0.1) 0.41 (16; 0.04) 0.52 (39; 0.03) 0.53 ( I t ; 0.04) Early swarm - (0) 0.47 (18; 0.04) 0.47 (45; 0.02) 0.45 (9; 0.04) Late swarm - (0) 0.34 (9; 0.05) 0.30 (39; 0.03) 0.29 (6; 0.08)

Cost of swarming (cal/h) - 0.39 0.51 0.48

* Wing length: I: 3.69-3.99mm; 11: 4.0-4.3mm; 111: 4.31-4.6; IV: 4.61-5.15.

into four ascending size classes. We then computed the average caloric amount of carbohydrates (nectar, other sugars and glycogen) available to individuals in each of these sub-groups (Table 1) . The smallest size group, while present in the morning and evening resting collections, was totally absent from the swarming samples. By sub- tracting the average concentration of calories in the late swarmers from that found in the early swarmers, and assuming that they had swarmed for 20min. we estimated the caloric cost per hour of flight (Table 1). The cost of swarming for the males in the smallest swarming size group (11) was markedly lower than in the other groups. However, possibly due to the few individuals in this group, this difference was not statistically significant at the 0.05 level (F=0.19; P = 0.82). For all size groups, we found that swarming for 40 min would consume well over 50% of the reserves available.

Discussion

To fuel flight, insects can metabolize stored lipids, carbo- hydrates or amino acids. In general, insects that routinely undertake flights of long duration, such as leidopterans and orthopterans, rely on lipid reserves. Conversely, in groups where flight is usually of short duration, carbo- hydrates are used as fuel (Wheeler, 1989). Our results indicate that sugar (probably glucose) and glycogen are the fuels for male An.freeborni flight. Lipids, which are used for resting metabolism, are not used to fuel flight.

These conclusions are consistent with the work of Nayar & Van Handel (1971) who studied the energy metabolism of Aedes sollicitans and Ae.taeniorhynchus tethered to a flight mill. They established that carbohydrates were used by both sexes to sustain flight and that lipids were not utilized. Furthermore, in the absence of a recent sugar meal, glycogen was the fuel of choice, and trehalose was not used. We find that equal proportions of sugar (probably glucose) and glycogen are used by swarming males (Fig. 3). This indicates that a regulatory mechanism, mobilizing reserves from both sources, is at work. Ziegler & Schultz (1986) suggest that in Manduca sexta changes in the concentrations of haemolymph sugars control the

activity of glycogen phosphorylase. A similar mechanism could be operating in swarming mosquitoes.

For An.freeborni males in the field, nectar sugars are not immediately available to fuel flight activities because they sugar-feed after swarming is concluded (Figs 1 and 2). The time of sugar-feeding in mosquitoes is species specific; some species sugar-feed during daylight, others are crep- uscular in their habits, and some nocturnal (Yuval, 1992). In a qualitative study, Reisen et al. (1986) observed that 93% of swarming Culex tarsalis males were negative to the cold anthrone test, which detects recent nectar feeding, while 71% of males captured in the early morning showed signs of a recent sugar meal. Our results reveal a similar pattern.

From an energetic perspective, sugar-feeding after flight activity may be considered maladaptive, as the storage and subsequent mobilization of energy reserves i s more costly than direct utilization of nectar (Wheeler, 1489). However, there may be energetic constraints on flying with a full crop, because a recent nectar meal contains as much as 60% water. In the field, we have observed that odonate predators are active during the hour before sunset, both in the air (Puntala and Erythemis sp.) and near flowering plants (lschnura sp.). An argument can be made for the evolution of sugar-feeding patterns that, though more expensive energetically, uncouple sugar-feeding from the activity of visually oriented predators.

Lipids were highly correlated to size and probably reflect reserves accumulated during larval development (Briegel, 1990a. b). Some of these lipids are tied up in various tissues, while others could (metaphorically), serve as an energetic nest egg, a trust fund which complements the cash account of trehalose and glycogen. Though not available to fuel flight, lipids are used in resting metab- olism. One question worth investigating is whether males that repeatedly fail to acquire a sugar meal can catabolize lipids into glucose or glycogen in order to be capable of flight.

The function of swarms in An.freeborni is reproductive, and the mating success of males depends on their size, when they join a swarm and how long they remain in it (Yuval et ul., 1993; Yuval & Bouskila, 1993). Males in our smallest size group had (on average) higher levels of

78 B. Yuval, M. L . Hol l iday -Hanson and R. K . . W a s h i n o

energy reserves than males in the other groups. However, these individuals never appeared in swarms (Table 1). Whether these males employ an alternative mating tactic, as do the smaller males in populations of some chironomid midge species (McLachlan & Neems, 1989), is still moot. Interestingly, males in the second smallest size group (the smallest males to swarm) began swarming with an average higher caloric content than found in the resting sample of the same size group. This suggests that males in this size group will not swarm if their energy reserves are below a certain level. Beyond these size-related thresholds, the amount of carbohydrates available may determine when a male joins a swam and certainly determine how long he will be able to sustain flight.

The levels of nectar sugars in males from the early morning samples indicate that not all individuals feed on sugar every night (Fig. 2). The lack of consistent correlation between sugar content and size indicates that success at acquiring a sugar meal is independent of size. The energetic cost of swarming relative to the average energy reserves of individual males indicates that this activity can consume over 50% of the energy available for flight (Table 1). We suggest that there is a strong link between the ability of an individual male to find and exploit a nectar source, and his lifetime reproductive success.

Acknowledgments

We thank Angela Silva for technical assistance, J. Rosenheini for comments on the manuscript, and S. S. Duffey for use of equipment and many patient discussions. Supported by University of California Mosquito Research Funds and a grant from The UNDP/World BanklW.H.0. Special Programme for Research and Training in Tropical Diseases (TDR) to B.Y.

References

Bricgcl, H. (l9Wa) Fecundity, metabolism and body size in Anopheles (Diptcra: Culicidae), vectors of malaria. Journal of Medical Entomology, 27, 839 -850.

Bricgcl, H. ( IYWb) Mctabolic relationship bctwccn female body sizc, rcscrvcs, and fecundity in Aedes aegypti. Journal of Insect Physiology. 36, 165-172.

Cootcr. R.J. (1989) Swarm flight behavior in flies and locusts. 1nsec.t Flight (ed. by G . J. Goldsworthy and C. H. Whceler), pp. 165-203. CRC Prcss. Boca Raton, Florida.

Flcckcr. A S . , Allan, J.D. & McClintock, N.L. (1988) Male body sizc and mating succcss in swarms of the mayfly Epeorus longimanus. Holurctic Ecology, 11, 280-285.

McLachlan. A. & Nccms, R. (1989) An alternative mating system in small male insects. Ec:ological Entomology, 14, 85-91.

Nayar, J.K. & Van Handel. E. (1971) The fucl for sustained mosquito flight. Journal of’lnsect Physiology, 17, 471 -481.

Nccms, R.M., Lazarus. J . & Mclachlan. A.J. (1992) Swarming behavior in malc chironomid midgcs: a cost- bcncfit analysis. Behavioral Ecology, 3, 285-290.

Nccms, R.M., McLachlan, A.J. & Chambers, R. (1990) Body sizc and lifetime mating succcss of male midges (Diptcra: Chironomidac). Animal Behavior. 40, 648-652.

O’Meara, G.F. (1987) Nutritional ecology o f blood feeding Diptera. Nutririonul Ecology of Insects, Mites unrl Spiders (ed. by F. Slansky, Jr and J. G. Rodriguez). pp. 741-764. John Wilcy & Sons, New York.

Parker, G.A. (1978) The evolution of compctitivc niatc searching. Annual Review of Entomology, 23, 176- 196.

Pctcrsson, E. (1989) Age-associated malc mating succcss i n three swarming caddis fly species (Trichoptcra: Lcptoceridac). Ecological Entomology, 14, 335-340.

Petersson, E. (1090a) Malc age, copulation duration and inscmi- nation success in Mystarides azurea (Lcptoceridae: Trichoptcra).

Petcrsson, E. (1990b) Age-assortativc mating patterns in two swarming caddisfly species (Trichoptcra: Lcptoccridac). Jruirnal of Insect Behavior, 3. 797-803.

Reiscn, W.K., Meycr. R.P. & Milby. M.M. (1986) Pattcrns o f fructose fceding by Cu1e.r tarsalis (Diptcra: Culicidac). Journul of Medical Entotnology. 23, 366-373.

Sullivan, R.T. (1981) Inscct swarming and mating. Florida Ento- mologist. 64, 44-65.

Svcnsson, B. & Pctcrsson, E. (1992) Why insects swarm: testing the models for lck mating systems on swarming Empis borealis fcmales. Behavioral Ecology and Sociobiologv, 31. 253-261.

Thornhill, R. (1980) Scxual selection within mating swarms of the lovcbug, Plecia neartica (Diptera: Bibionidac). Animal Behavior, 28, 405-412.

Van Handel. E. (1985) Rapid dctcrmination of total lipids in mosquitoes. Journal of the Americun Mosquito Control Associ- ation, 1, 302-304.

Van Haiidcl. E. & Day. J.F. (19YO) Nectar-fccding habits of Aedes taeniorhynchus. Journul of the Americun Mosquito Control Association. 6 , 270-273.

Wheclcr, C.H. (1989) Mobilization and transport o f fuels to the flight muscles. Insect Flight (ed. by G . J . Goldsworthy and C. H. Wheeler), pp. 273-303. CRC Press. Boca Raton. Florida.

Yuval, B. (1992) The other habit - sugar fceding by mosquitoes. Bulletin of the Society for Vector Ecology, 17, 150-156.

Yuval, B. & Bouskila, A. (1993) Tcmporal dynamics of mating and predation in mosquito swarms. Oecologiu, 95, 65--6Y.

Yuval, B.. Wekcsa, J.W. & Washino, R.K. (1993) Effects of body sizc on swarming behavior and mating succcss of inalc Anopheles freeborni (Diptera: Culicidac). Journal of Insect Behavior. 6 , 333-342.

Zar, J.H. (1984) Biostatistical Analysis. Prciiticc Hall, New Jcrscy. Zicgler. R. & Schultz. M. (1986) Rcgulatioii of carbohydrate

metabolism during flight in Manduca sextu. Journal of 1nsec.t Pkysiology, 32, 997- 1OOI.

Ethology, 85. 156-162.

Acceptcd 7 August 1993