The larvae of Spodoptera litura were reared on an artificial diet, and the flight capability, and triacylglycerol (TG) level plus its fatty acid composition in 3-day-old sexually mature and non-fed adults were compared. In males, during 3 hr of tethered flight, the levels of abdominal TG and its fatty acid components did not change. But thereafter the TG and fatty acids, significantly unsaturated fatty acids in TG declined in their levels with the prolongation of flight, unsaturated fatty acids being exhausted preceding saturated fatty acid decline. When males were tested by tethered flight for 20 hr, some could fly for nearly the whole period, and were judged to be able to fly for approximately 24 hr, depending on the level of residual TG. Fatty aids in TG decreased in females similarly to males during tethered flight and some females with fully developed ovaries deposited eggs after 12 hr of flight similarly to non-flown individuals, which supports the long-distance flight capability even in sexually mature females. These results are discussed with regard to the overseas migration of this moth.
In long-distance migratory insects, triacylglycerol (TG), a neutral lipid which is reserved in the fat body and transported to flight muscles in the hemolymph as diacylglycerol (DG) bound to lipophorin (Chino, 1985; Soulages and Wells, 1994; Ryan and Van der Horst, 2000), has generally been accepted to be utilized as the major flight fuel (Beenakkers et al., 1985: Blacklock and Ryan, 1994). However, only a few studies have been reported on the storage and utilization of TG and its fatty acid composition in long-distance migratory insects (Van Handel, 1974; Van der Horst et al., 1980; Teo et al., 1987; Zongshun and Ouyang, 1995).
Adult moths of the common cutworm Spodoptera litura, are regarded to migrate overseas, because males have been caught on weather-forecasting ships on oceans (Asahina and Tsuruoka, 1968), and sudden and coincidental occurrences of these moths have often been recorded in two locations 180 km apart at the time of a typhoon approach to these sites (Murata et al., 1998). Spodopera litura does not enter diapause in any of its developmental stages (Ishida and Miyashita, 1976), and this supports immigration of the moths overseas to Kyushu, the southernmost mainland of Japan, where the temperature is sometimes less than 0°C in winter but outbreaks of larvae are often recorded. One of the observations inconsistent with their long-distance migration is the early development of ovaries in females who can lay eggs within several days after adult emergence (Okamoto and Okada, 1968).
In this species, activities related to the flight have been examined by tethered flight (Oyama, 1976; Noda and Kamano, 1988) and by actographs (Kawasaki, 1986; Saito, 2000a) to determine the effect of sex and aging, without checking the flight fuel reserves.
In this study, depending on these knowledge, we tried to elucidate how the TG level and its fatty acid composition change during tethered flight in S. litura adults of both sexes at the time of high reproductive activity to estimate their flight capability. We demonstrate that fatty acids in TG are the major source of flight energy in the adults, and that even the females with mature ovaries are able to migrate and lay eggs similarly to non-flown females.
MATERIALS AND METHODS
Spodoptera litura eggs were kindly supplied by Sumitomo Chemical Co. Ltd. Their progenies were reared at 25°C under a 16 hr L-8 hr D photo-regime for several generations on an artificial diet composed mostly of kidney beans, wheat germ and yeast powder (Okamoto and Okada, 1968). The larvae for the experiments were reared on the artificial diet under the same conditions used for the stock culture. After the ecdysis to the sixth (last) larval stadium, 50 larvae were transferred in a cage (23 cm×33 cm, 6 cm h) and reared to adults. Adult emergence was checked every 8 hr and, to avoid wasting the flight energy source, each adult was maintained in a test tube (1.2 cm i.d., 10.5 cm h) containing a segment of filter paper moistened with water, which was exchanged every other day, until the flight experiment.
Each 3-day-old moth (72–80 hr after emergence) was anesthetized under carbon dioxide, and the scales on the back of thorax were removed using a small brush. The animal was then anesthetized again, and a polyethylene tube (d. 2.5 mm) with a pin inserted from the other end (pinhead inside the tube and tip outside) was adhered to the back of the thorax where the scales had been removed, with the aid of manicure mixed with small glass beads. After drying the adhered portion under carbon dioxide flow, the adult was suspended just below by piercing a Styrofoam plate with the tip of the pin. After recovery from anesthesia, the animal was allowed to fly. The flights of ten moths were measured for each set of experiments and recorded for 2 s every 30 s during 15 to 20 hr by a video camera. The flying times of each moth were counted by eye from the video film taken every 30 s, and the values in seconds were multiplied by 0.25 to obtain the total flight duration in minutes. If an adult did not start flying during the first 30 min, the leg was briefly touched with a small brush to insist on the flying. After flight, the abdomen was excised from the moth, frozen at −20°C, lyophilized within a week and stored at −20°C for lipid analysis. During the period of storage and lyophilization, slight changes in the lipid quantity and quality occurred, but were not to a degree which would cause significant effects on the interpretation of the results (data not shown). When a moth did not initiate flying during the course of flight experiment, even after the brief touch, it was removed from the set and frozen as a non-flyer. For non-flying controls, 0-, 3-, 7-and 10-day-old moths maintained in test tubes with water were treated similarly to the flown moths. To investigate the possible transport of lipid to the ovaries from other parts of the body, abdomens (including ovaries) or ovaries were taken from 0- and 3-dayold non-flown females and examined for TG levels.
Extraction of total lipid, TG separation and its fatty acid composition analysis were performed as described in a previous paper (Itoyama et al., 1999). Briefly, the lyophilized abdomens or ovaries were weighed, and the total lipid was extracted with chloroform-methanol (2:1, v/v) according to Folch et al. (1957). The concentrated extract was fractionated by thin-layer chromatography on a silica gel G plate (Whatman K5, 20 cm×20 cm) in petroleum ether-diethylether-acetic acid (82:18:1, v/v/v) as the solvent system and the TG fraction was cut from the plate. The fatty acids in the fractions were converted to their methyl esters, and their fatty acid compositions were analyzed by gas-liquid chromatography (GLC, Shimazu GC-14B, Kyoto, Japan) on a fused silica capillary column (HR-SS-10, i.d. 0.25 mm×25 m, Shinwa Chemical Industries Ltd.), using helium as the carrier gas. The oven temperature for analysis was fixed at 170°C. The amount of TG was represented as the total μmoles of fatty acids recovered in the fraction per gram of dry weight of insects (μmol/g d.w.). The values were corrected by comparison to the recovery of pentadecanoic acid as an internal standard.
Statistical analyses in this article were performed with Stat View 4.0 (SAS Institute, 1990).
Changes in TG levels and their fatty acid compositions during tethered flight in males
The TG fatty acid level per g dry weight in the abdomen of 3-day-old males was 850 μmol on average, and significantly declined with prolongation of tethered flight (r=−0.723, P<0.0001), to 409 μmol after 15 hr of flight, and 114 μmol after flight longer than 18 hr (Fig. 1). In contrast, in non-flown moths maintained without food, the TG fatty acid level declined far more slowly but still significantly (P=0.0012), and 549 μmol still remained in the abdomen even at 10 days after emergence (Fig. 1). Hourly loss of TG fatty acid per g dry weight was 39 μmol in the flown males, but was only 1.4 μmol in the non-flown ones, i.e., 28-times more TG fatty acid consumption during flight than when at rest.
Fatty acid levels in abdominal TG in 3-day-old flown male moths were shown in Table 1. Comparisons were also done in the non-flown males kept without food for a further 7 days (Table 2), where relative quantities of each fatty acid per g dry weight are represented as ratios to the value in 3-day-old males; to make comparison easier with the flown ones of this age. In the abdominal TG of flown and non-flown males (Tables 1 and 2), palmitic (C16:0) and oleic (C18:1) acids were the predominant fatty acids, followed by palmitoleic (C16:1) and linoleic (C18:2) acids, with minor components of linolenic (C18:3), stearic (C18:0), miristic (C14:0) and lauric (C 12:0) acids. During 3 hr of tethered flight, the levels of all fatty acids in abdominal TG did not significantly change, and so TG fatty acid level was regarded not to decrease during this period, although the correlation between TG level and flight duration was calculated in Fig. 1 assuming continuous consumption of TG during flight. But thereafter all of the four unsaturated fatty acids significantly decreased with prolongation of flight (ANOVA, linolenic acid: P<0.0001; linoleic acid: P<0.0001; oleic acid: P=0.0001; palmitoleic acid: P<0.0001). The level of a predominant saturated fatty acid, palmitic acid decreased rapidly as flight duration exceeded 15 hr, although the level did not significantly change when the moths flied less than 15 hr (ANOVA, P=0.0276) (Table 1). On the other hand, the levels of other three saturated fatty acid did not significantly change with flight duration (ANOVA, stearic acid (C 18:0): P=0.1336; miristic acid (C 14:0): P=0.9777; lauric acid (C 12:0): P=0.3445) (Table 1).
Changes in fatty acid quantity (mmol / g. d. w.) of TG in the abdomen of S. litura males during tethered flight1,2
Changes in fatty acid quantity (mmol / g. d. w.) of TG in the abdomen of S. litura males under non-flown condition1,2
In the abdominal TG of non-flown males (Table 2), the levels of the four unsaturated fatty acids significantly decreased with age (ANOVA, linolenic acid (C 18:3): P=0.0030; linoleic acid (C 18:2): P=0.0016; oleic acid (C18:1): P=0.0034; palmitoleic acid (C16:1): P=0.0003). The level of stearic acid (C18:0) decreased in 3 days after emergence, but did not change thereafter. On the other hand, the other three saturated fatty acids did not change (ANOVA, palmitic acid (C 16:0): P=0.1128; miristic acid (C 14:0): P=0.9138; lauric acid (C 12:0): P=0.3124) (Table 2).
Flight capability, TG content and fecundity in flown females
The wet weight of the ovaries in non-flown female adult increased in 3 days after emergence (ANOVA, P<0.001) (Fig. 2). In this period, TG in the abdomen including ovaries did not change (U-test, P>0.05), but the TG level in the ovaries per g dry weight increased from 148 μmol, i.e., 24% of the total TG in the abdomen at emergence, to 566 μmol, i.e., 88% of the total abdominal TG (U-test, P<0.05) (Fig. 3). The proportion of unsaturated fatty acids in the abdominal TG fatty acids slightly changed during the first three days of adult development (from 37 to 44%), but the proportion in ovarian TG' greatly changed (from 4% at day 0 to 48% at day 3) (Fig. 4).
The TG content per g dry weight of the abdomen in females decreased with prolongation of flight, from 672 μmol in non-flown to 198 μmol after 12 hr of flight (r=−0.649, P=0.0026); the average loss of TG per g dry weight per hr being 37 μmol (Fig. 5A). When females mated after tethered flight, the number of eggs they deposited significantly declined with the increase of flight periods (r=−0.493, P=0.0168), but half of the females could lay eggs even after 12 hr of flight in quantities similar to some of the non-flown controls (Fig. 5B).
Utilization of fatty acids in TG in long flights
In long-distance migratory insects, a rapid decline in carbohydrate followed by an increase in lipid (diacylglycerol) in the hemolymph during flight have been generally accepted to indicate the utilization of carbohydrate for initial flight, and then lipid for sustained flight (Van der Horst et al., 1980; Teo et al., 1987). However, virtually no studies have been published on the fluctuations of even lipid levels in the whole body or fat body, the main reserve organ of lipids, during long flights near exhaustion except for the following data: 40–58% decrease in lipid during 16–32 hr of tethered flight in Spodoptera frugiperda, and 34–85% decrease in lipid during 23–40 hr of tethered flight in Heliothis zea (Van Handel, 1974).
Here, we demonstrated that the TG fatty acid level in the abdomen of 3-day-old male S. litura moths declined with prolongation of tethered flight, at a rate of 39 μmol per g dry weight per hr, being 28 times higher compared to non-flown starved males (Fig. 1). In females, the average loss of TG per g dry weight per hr was 37 μmol (Fig. 5A). But, restricting to the changes during 3 hr of flight, the TG fatty acid level did not significantly decrease as being supported by non-significant declines of any TG fatty acids (Table 1). This seems to indicate that TG fatty acids were not consumed during early flight, and may suggest initial utilization of carbohydrates as flight fuel, as being demonstrated in other long-distance migratory insects (Van der Horst et al., 1980; Teo et al., 1987).
During flight from 3 hr up to 15 hr, the levels of all unsaturated fatty acids in the TG significantly decreased, while those of all saturated fatty acids did not change and rather seem to increase except palmitic acid (Table 1). In non-flown males, the four unsaturated fatty acids in TG declined from 3 to 7 days of starvation, while a decrease in quantity was not observed in any of saturated fatty acids (Table 2). So, the utilization of respective TG fatty acids seems to proceed similarly during starvation and flight, but to be highly enhanced by flight. As the level of respective fatty acid reflects the difference between the synthesis and consumption, the present result may indicate that synthesis and consumption equally proceeded in saturated fatty acids, while consumption overwhelmed synthesis in unsaturated fatty acid.
As the moths used for tethered flight were maintained only on water, so all materials with carbon skeleton should be supplied by themselves. One of resources for fatty acid synthesis will be carbohydrate, but carbohydrates would be exhausted if these were the major fuels during early flight as mentioned above. Other possible sources for the synthesis will be from inter-conversion of saturated and unsaturated fatty acids, but metabolic conversion between them during degradation by β-oxidation have not known (Conn et al., 1987). So, it may be also possible to explain that the decline of respective fatty acids in TG was mostly due to consumption of them and so, the present data may indicate utilization of unsaturated fatty acids preceding to that of saturated fatty acids in flight longer than 3 hr. More detailed studies are required to conclude either both saturated and unsaturated were utilized equally or selectively during tethered flight.
In other long-distance migratory insects, a change of unsaturated fatty acid level during flight has been reported, e.g., in Mythimna separata, oleic acid decreased in the fat body and increased in the flight muscle during tethered flight (Zongshun and Ouyang, 1995). In Cnaphalocrosis medinalis, linolenic acid in TG seemed to be utilized first because migrants contained TG with essentially no linolenic acid, while non-immigrants had a high level of linolenic acid in TG (Murata and Tojo, 2001). In the migratory locust Schistocerca gregaria, the ratio of unsaturated fatty acids in TG' is kept over 60% during one month of feeding in adulthood, supporting the hypothesis that unsaturated fatty acids are the major flight fuel, although analyses of adults after long flights were not conducted (Schneider and Dorn, 1994).
Flight capability of reproductively mature moths
In S. litura, the females release sex pheromones within one or two days after emergence, and the males become able to respond to the pheromones a little later (Yushima et al., 1973). Since the females used in the present experiment matured ovaries within 3 days of emergence (Fig. 2), both the 3-day-old males and females used in the tethered flight could be said to be sexually mature. Here, we demonstrated that even these sexually mature males and females could fly for over 18 and 12 hr, respectively, in tethered flights, although they had been maintained without food for 3 days (Figs. 1 and 5), supporting their potential to undertake long-distance migration. It seems likely that they are able to fly longer if they were given food during these three days. Since females lost nearly two-thirds of their TG during 12 hr of flight, but still maintained a high level of TG (Fig. 5) similar to the 12 hr-flown males (Fig. 1), they would be able to fly for several more hours. In males which were flown for more than 18 hr, the abdominal TG per g dry weight was 144 μmol (Fig. 1), while the level was approximately 50 μmol in those moths caught at the time of a typhoon approach, which were expected to have immigrated overseas on the typhoon winds (Murata, 2001). Judging from the decrease of abdominal TG in males during tethered flight (Fig. 1), the males caught at the time of the typhoon approach were estimated to have flown for nearly 24 hr (Murata, 2001). In such cases, saturated fatty acids would be utilized for the flight activity. The reason why the unsaturated fatty acids in TG are utilized initially as the flight energy source before the saturated acids remains unclear, although it might be due to the presence of specific enzymes necessary for the isomerization and oxidation of the unsaturated fatty acids in muscles (Conn et al., 1987)
We showed a prominent accumulation of TG in the ovaries during three days of ovarian development with yolk deposition. During this period, the proportion of unsaturated fatty acid in abdominal TG's remained 40% (Fig. 4). This suggests the increase of TG fatty acids in the ovaries being mostly due to the uptake of these fatty acids, possibly bound with vitellogenin, by the ovaries, as have been shown in many insect species (Kunkel and Nordin, 1985; Raikhel and Dhadialla, 1992). In fact, vitellogenin with 9% of lipid content in S. litura first appears in the hemolymph one day before adult emergence, and prominently increases the concentration shortly after emergence (Maruta et al., 2002), preceding the yolk deposition, i.e., vitellogenin uptake by oocytes (Fig. 2). When 3-day-old females were flown, 37 μmol of abdominal TG fatty acids per g dry weight was consumed per hr as in the flown males (39 μmol). It is reasonable to assume that the TG deposited in the yolk of the ovaries in 3-day-old females would become the major source of flight energy, since 88% of the TG was distributed in the ovaries at the time of flight initiation (Fig. 4). Absorption of oocytes is known in many insects, when they are starved or not inseminated (Chapmann, 1998), but virtually no studies have been published on the possible utilization of the fatty acids deposited in the oocytes as an energy resource for the flight muscles.
We further showed that some sexually mature females laid a similar number of eggs to the non-flown ones, even after 12 hr of flight (Fig. 4). The S. litura females in sexually immature stages show similar high flight activity to those in mature stages (data not shown). These results seem to coincide with those obtained by actographs in which the moths were allowed to fly freely: females of S. litura actively flew at night for several days, and males were more active for longer periods (Saito, 2000a). This would be an advantageous characteristic for the survival of a migratory insect: females first migrate and then reproduce. In a long-distance migratory cotton bollworm Helicoverpa armigera, the females have mature eggs within three days after emergence as in S. litura, and showed nearly ten days of active flight activity by actographs, although copulation significantly reduced their flight activity (Saito, 2000b). Studies are now in progress to see whether or not preceding copulation has any effect on the long-distance migratory capability of S. litura females.
Noda and Kamano (1988) demonstrated that, during 10 hr experiments with a flight mill, 3-day-old females and males of S. litura flew for only 3 or 4 hr, and estimated that they would be able to fly less than 100 km in a night, thus denying a long flight capability of this species. In another tethered flight experiment, S. litura could fly only for 10 min at the maximum (Oyama, 1976). Although such contradictory results might be due to the different methods applied to check the flight ability, the results seem to suggest genetic differences in the migratory potentials of S. litura populations, as has been observed in several species of insects (for a review: Gatehouse, 1997). We are finding such variations among field-collected S. litura and these data will be published elsewhere.
Recent trajectory analyses of 850 hPa air masses with strong winds of over 10 m/s and records of moths trapped in pheromone traps strongly suggest the overseas immigration of S. litura to Japan in spring and autumn rainy seasons within 24 hr from mainland China and the Korean Peninsula (Murata, Inoue, Cheng and Tojo, unpublished). Our present data support the hypothesis that their lipid reserve is sufficient for long flights, and show that they have a high flight capability in no-wind conditions for the period nearly corresponding to the duration estimated to be necessary for immigration to Japan with the aid of air masses at 850 hPa. In the case of wind-born migration, the moths can be expected to migrate or disperse for longer distances than those estimated in laboratory experiments.
We would like to thank Makoto Hatakoshi of Sumitomo Chemical Co. Ltd. for supplying the Spodoptera litura eggs. This work was partly supported by a Grant-in-Aid from the Japan Society for Promotion of Science (No. 13460023).
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