Enhancing offspring quality or quantity? Different ways for using nectar amino acids in female butterflies

Butterfly-pollinated flowers offer nectar with higher amino acid concentrations than most flowers pollinated by other animals, and female butterflies of some species prefer to consume amino acid-rich nectar. However, for over 30 years, there has been an ongoing discussion about whether nectar amino acids benefit butterfly fitness. A clear positive effect was only shown for the nectar-feeding Araschnia levana, and females of the fruit-feeding Bicyclus anynana also increased offspring quality when they were fed amino acids as adults. Thus, severe doubts remain about the general significance of these single positive results. We therefore tested a further species from a phylogenetically different butterfly subfamily, the small heath (Coenonympha pamphilus L., Satyrinae), taking into account feeding conditions over the whole life cycle of this species. C. pamphilus females receiving nectar amino acids as adults, irrespective of larval food quality, produced heavier larvae and also increased the hatching success of their eggs over the oviposition period. Furthermore, females raised under nitrogen-poor larval conditions tended to use nectar amino acids to increase the number of eggs laid. Thus, C. pamphilus females used nectar amino acids primarily to increase their offspring quality, and secondly tended to increase offspring quantity, if larval resources were scarce, showing a resource allocation pattern differing from both B. anynana and A. levana. Our study supports the old postulate that nectar amino acids generally enhance butterfly fitness.     

Amino acids in nectar enhance butterfly fecundity: a long-awaited link

Thirty years ago, researchers discovered that flowers pollinated by butterflies are consistently rich in nectar amino acids, and more recent findings have shown that butterflies prefer nectar with high amino acid content. These observations led to speculation that amino acids in nectar enhance butterfly fitness and that butterflies have acted as agents of natural selection on nectar composition. Despite a number of experimental efforts over the years, convincing proof that nectar amino acids affect butterfly fitness has been lacking. Here, we provide the first evidence that amino acids in nectar have a positive effect on fecundity of one butterfly species, supporting the existence of a relationship between nectar preferences and fitness benefits. Map butterflies (Araschnia levana L.) raised under natural larval food conditions laid more eggs when they were fed nectar containing amino acids, whereas nectar amino acids had no effect on the number of eggs laid by butterflies raised on larval food rich in nitrogen. Uptake and utilization of nectar amino acids by map butterflies appear to be compensatory mechanisms enabling them to override impacts of poor larval food. These results provide strong support for the long-standing postulate that nectar amino acids benefit butterflies.     

Introduced fire ants can exclude native ants from critical mutualist-provided resources

After over 30 years of research, it was recently shown that nectar amino acids increase female butterfly fecundity. However, little attention has been paid to the effect of nectar amino acids on male butterfly reproduction. Here, we show that larval food conditions (nitrogen-rich vs. nitrogen-poor host plants) and adult diet quality (nectar with or without amino acids) affected the amount of consumed nectar in Coenonympha pamphilus males. Furthermore, amino acids in the nectar diet of males increased progeny’s larval hatching mass, irrespective of paternal larval reserves. Our study takes the whole reproductive cycle of male butterflies into account, and also considers the role of females in passing male nutrients to offspring, as males’ realized reproduction was examined indirectly via nuptial gifts, by female performance. With this comprehensive approach, we demonstrate for the first time that nectar amino acids can improve male butterfly reproduction, supporting the old postulate that nectar amino acids generally enhance butterfly fitness.     

Mating frequency influences nectar amino acid preference of Pieris napi

It is generally assumed that butterflies, as is the case with many holometabolous insects, rely primarily on nutrients gathered by larval feeding for somatic maintenance and fecundity. These reserves can be supplemented by adult feeding and in some cases by nuptial gifts passed from the males to the females during mating. Recent findings indicate that female butterflies detect and prefer nectar with high levels of amino acids, thus calling new attention to this nutritive source. Polyandrous species can further supplement their larval stores with additional nuptial gifts. This study examined how mating frequency of the polyandrous butterfly Pieris napi affects the female's preference for nectar amino acids. Females of this species generally detect and prefer nectar mimics containing amino acids. However, nectar amino acid preference is significantly lower in mated females. Furthermore, nectar amino acid preference increases when females are not allowed to remate, whereas the preference of twice-mated females remains constant at a lower level. These results indicate a versatile response of females to nectar amino acids, depending on their nutritional status; they may even switch their source of amino acids between adult feeding and nuptial gifts.     

Amino acid sources in the adult diet do not affect life span and fecundity in the fruit-feeding butterfly Bicyclus anynana

Abstract.  1. In tropical forests, the adults of many butterfly species feed on fruits rather than nectar from flowers and have long life spans. Rotting fruit and nectar differ from each other in many respects, including sources of amino acids and microbial life. If amino acids in the adult diet can be used for reproduction, this may have facilitated the evolution of extended life spans in this guild.
2. This issue was addressed by investigating effects of banana, yeast, and amino acids in the adult diet of the fruit-feeding butterfly Bicyclus anynana (Lepidoptera) on longevity and female reproductive output in two experiments.
3. Results showed that in the fruit-feeding butterfly B. anynana : (i) banana juice, but not sliced banana or added amino acids extend life span compared with a sugar solution of similar composition; (ii) compared with this sugar solution, other cohorts (banana juice-amino acid enriched) did not have significantly higher reproductive outputs; (iii) yeast does not represent a valuable source of nutrients; (iv) caloric restriction may cause decreased life span and rate of reproduction; and (v) increased rates of reproduction have a life span cost.     

Nectar and hostplant scarcity limit populations of an endangered Oregon butterfly

As grassland habitats become degraded, declines in juvenile and adult food resources may limit populations of rare insects. Fender's blue butterfly (Icaricia icarioides fenderi), a species proposed for listing as endangered under the US Endangered Species Act, survives in remnants of upland prairie in western Oregon. We investigated the effects of limited larval hostplants and adult nectar sources on butterfly population size at four sites that encompass a range of resource densities. We used coarse and detailed estimates of resource abundance to test hypotheses relating resource quantity to population size. Coarse estimates of resources (percent cover of hostplant and density of nectar flowers) suggest that butterfly population size is not associated with resource availability. However, more detailed estimates of resources (density of hostplant leaves and quantity of nectar from native nectar sources) suggest that butterfly population size is strongly associated with resource availability. The results of this study suggest that restoring degraded habitat by augmenting adult and larval resources will play an important role in managing populations of this rare butterfly. Received: 20 June 1998 / Accepted: 25 November 1998     

Preference of cabbage white butterflies and honey bees for nectar that contains amino acids

Summary Amino acids occur in most floral nectars but their role in pollinator attraction is relatively unstudied. Nectars of butterfly-pollinated flower tend to have higher concentrations of amino acids than do flowers pollinated by bees and many other animals, suggesting that amino acids are important attractants of butterflies to flowers. In order to determine whether amino acids are important in attracting butterflies and bees, we tested the preference of cabbage white butterflies (Pieris rapae) and honey bees (Apis mellifera) by allowing them to feed from artificial flowers containing sugar-only or sugar-amino acid mimics ofLantana camara nectar. Honey bees and female cabbage white butterflies consumed more sugar-amino acid nectar than sugar-only nectar. In addition, female cabbage white butterflies visited artificial flowers containing sugar-amino acid nectars more frequently than flowers containing sugar-only nectars; honey bees spent more time consuming the sugar-amino acid nectar. Male cabbage white butterflies did not discriminate between the two nectars. These results support the hypothesis that the amino acids of nectar contribute to pollinator attraction and/or feeding.     

Can nectar properties explain sex‐specific flower preferences in the Adonis Blue butterfly Lysandra bellargus?

1. Field observations in the Swiss Jura mountains showed that males and females of the bivoltine Adonis Blue butterfly Lysandra bellargus Rott. differed significantly in their flower visitation patterns. 2. In both generations, females visited a broader range of available nectar plants than did males. The specific flower visitation patterns of males and females were not affected by the general availability and abundance of potential nectar plant species during both flight periods, indicating high selectivity for nectar plants by both males and females. 3. In addition, the sexes differed in their nectar foraging behaviours: distances between successively visited flowers were significantly longer in males than in females, indicating that male and female butterflies have different foraging strategies. 4. Investigations of nectar characteristics showed that the sexes preferred flowers with different nectar compositions. Males of both generations preferred flowers with high proportions of sucrose and high amounts of total sugar, whereas females preferred flowers with high portions of glucose in their nectar, and, in the spring generation, flowers rich in amino acids. 5. Flowers visited exclusively by males or females in spring differed significantly in their amino acid composition. 6. This clear‐cut pattern did not hold for the autumn generation, most probably due to the limited availability of flowers. 7. The observed nectar foraging patterns underline the importance of adult feeding for longevity and reproduction in butterflies. The findings are particularly relevant for conservation, because L. bellargus is an increasingly threatened species in many European countries.     

The amino acids used in reproduction by butterflies: a comparative study of dietary sources using compound-specific stable isotope analysis

It is a nutritional challenge for nectar-feeding insects to meet the amino acid requirements of oviposition. Here we investigate whether egg amino acids derive from larval diet or are synthesized from nectar sugar in four species of butterfly: Colias eurytheme, Speyeria mormonia, Euphydryas chalcedona, and Heliconius charitonia. These species exhibit a range of life history and differ in degree of shared phylogeny. We use 13C differences among plants to identify dietary sources of amino acid carbon, and we measure amino acid 13C using compound-specific stable isotope analysis. Egg essential amino acids derived solely from the larval diet, with no evidence for metabolic carbon remodeling. Carbon in nonessential amino acids from eggs derived primarily from nectar sugars, with consistent variation in amino acid turnover. There was no relationship between the nonessential amino acids of eggs and host plants, demonstrating extensive metabolic remodeling. Differences between species in carbon turnover were reflected at the molecular level, particularly by glutamate and aspartate. Essential amino acid 13C varied in a highly consistent pattern among larval host plants, reflecting a common isotopic "fingerprint" associated with plant biosynthesis. These data demonstrate conservative patterns of amino acid metabolism among Lepidoptera and the power of molecular stable isotope analyses for evaluating nutrient metabolism in situ.     

The extremely long-tongued neotropical butterfly Eurybia lycisca (Riodinidae): proboscis morphology and flower handling

Few species of true butterflies (Lepidoptera: Papilionoidea) have evolved a proboscis that greatly exceeds the length of the body. This study is the first to examine the morphology of an extremely long butterfly proboscis and to describe how it is used to obtain nectar from flowers with very deep corolla tubes. The proboscis of Eurybia lycisca (Riodinidae) is approximately twice as long as the body. It has a maximal length of 45.6?mm (mean length 36.5?mm?±?4.1?S.D., N?=?20) and is extremely thin, measuring only about 0.26?mm at its maximum diameter. The proboscis has a unique arrangement of short sensilla at the tip, and its musculature arrangement is derived. The flower handling times on the preferred nectar plant, Calathea crotalifera (Marantaceae), were exceptionally long (mean 54.5?sec?±?28.5?S.D., N?=?26). When feeding on the deep flowers remarkably few proboscis movements occur. The relationship between Eurybia lycisca and its preferred nectar plant and larval host plant, Calathea crotalifera, is not mutualistic since the butterfly exploits the flowers without contributing to their pollination. We hypothesize that the extraordinarily long proboscis of Eurybia lycisca is an adaptation for capitalizing on the pre-existing mutualistic interaction of the host plant with its pollinating long-tongued nectar feeding insects.     

The Relationship Between Habitat Selection and Preference for Adult and Larval Food Resources in the Polyphagous Butterfly <Emphasis Type="Italic">Vanessa cardui</Emphasis> (Lepidoptera: Nymphalidae)

Search strategies can have profound fitness-effects for plant-feeding insects. Here I focus on the potential conflict between searching for nectar plants and for larval food plants. I test if the butterfly Vanessa cardui, which can use some of its larval food plants as nectar sources, is able to rationalize this search problem by combining the two search tasks. Lab-experiments revealed a higher oviposition preference for Cirsium arvense over Urtica dioica and a corresponding difference in larval performance. Contrary to predictions, there was no effect of inflorescences on oviposition. However, experiments in large outdoor cages showed a higher occupancy and a higher level of oviposition in patches with access to nectar sources, even on U. dioica. Hence, while there was no preference for individual plants with flowers, the results suggests that V. cardui is simplifying its search task to primarily search for hosts in nectar-rich patches. This strategy allows females to increase oviposition rate, but it is likely that it comes at the expense of not always using optimal host plants in terms of offspring performance.     

Nectar sugar and amino acid preferences of Battus philenor (Lepidoptera, Papilionidae)

Abstract.

• 1 Butterflies of Battus philenor were tested for their preferences for nectar sugars and amino acids in an outdoor cage experiment.
• 2 The butterflies clearly preferred both sucrose and fructose over glucose. They also preferred sucrose over fructose.
• 3 No other preferences were found to be statistically significant, although male butterflies tended to prefer a plain sugar solution over a sugar solution containing a mixture of amino acids: females consumed both of these solutions in almost equal proportions.
• 4 The results are discussed with respect to nectar composition of butterfly pollinated flowers, flower preferences, physiological and reproductive aspects of butterflies.

     

COMPARATIVE REPRODUCTIVE BIOLOGY OF HERMAPHRODITIC AND MALE-STERILE IRIS DOUGLASIANA HERB. (IRIDACEAE)

A comparative study of the reproductive biology of male-sterile and hermaphroditic plants in a gynodioecious population of Iris douglasiana Herb. (Iridaceae) was conducted at the University of California's Marine Laboratory at Bodega Bay, California, between 1976–1979. Each year of the study, there were 11.1% male-sterile plants in the population, some of which began blooming at the same time as the earliest blooming hermaphrodites. Male-sterile flowers made up between 7–21% of the flowers produced during the male-sterile flowering period. Male-sterile flowers had smaller sepals and petals than hermaphrodites, there were fewer of them per square meter, and they had fewer pollinated stigmas than did hermaphroditic flowers. In a test to determine pollinator preference, intact hermaphroditic flowers tended to have more pollinated stigmas than did hermaphrodites with their stamens removed or those flowers with shortened sepals made to resemble the smaller male-sterile flowers. Floral phenology and nectar-flow patterns were similar in both types of flowers as were the kinds of amino acids and sugar rewards in the nectar. Male-sterile flowers, however, produced much less nectar per flower. There were no significant differences in the number of ovules per flower or the number of seeds produced per capsule between the two flower types, but the loss of seeds through larval predation was much greater in capsules from hermaphroditic flowers. Early flowering and setting of seed by plants with male-sterile flowers could give them a reproductive advantage over plants with hermaphroditic flowers which experience higher levels of larval predation later in the growing season.     

The effects of insect visitors on nectar constituents in temperate plants

Summary The floral nectar of angiosperms is primarily a solution of simple sugars, but contains detectable amounts of other solutes, particularly amino acids. These have been regarded as diagnostic, for phylogenetic and taxonomic purposes, and their mean concentrations may be significant in relation to the pollination syndrome of a plant species. However, in several temperate flowers the amino acid concentration varied by an order of magnitude when measured at intervals through a single day. In open, cup-shaped flowers, this was partly due to post-secretory equilibration with the varying humidity of the air. But the effects of floral visitation by insects on nectar amino acids were also important, both in open flowers and in those with long corollas and more enclosed nectar. Visitors could add amino acids to nectar by direct contact, by salivation, by damaging the neighbouring tissues causing cell leakage, and by dislodging pollen into the nectar; the importance of each of these effects varied according to corolla morphology and the spectrum of visitors.     

Can Inachis io detect nectar amino acids at low concentrations?

Abstract This study tested Inachis io L. butterflies' preference for nectar amino acids using a dilution series (100, 75, 50, 25 and 0%) of the amino acid concentration of Lantana camara to determine how sensitive the insects are to low concentrations of amino acids, such as those found in the flowers of many plant species. Female butterflies clearly detected and preferred nectar amino acids even at the lowest concentration offered, whereas males did not. These results further support the hypothesis that female butterflies detect and select for nectar with amino acids at levels found in the field.     

Pollen feeding in the butterfly Heliconius charitonia: isotopic evidence for essential amino acid transfer from pollen to eggs

Heliconius and Laparus butterflies exhibit a unique pollen-collecting behaviour that enhances lifespan and fecundity. The specific nutritional contribution of pollen, however, had not been previously demonstrated. We used stable isotope variation to trace the carbon flow into eggs from corn pollen provided experimentally to ovipositing female Heliconius charitonia, and to evaluate the use of isotopically contrasting nectar sugars in egg amino acids. The delta(13)C of individual amino acids from pollen, larval host plant and the eggs from experimental butterflies was measured with gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS), to evaluate amino acid transfer. The delta(13)C of egg essential amino acids indicated a transfer of essential amino acids from pollen to butterfly eggs. However, the delta(13)C of non-essential amino acids reflected the isotopic composition of the artificial nectar, indicating that H. charitonia synthesizes non-essential amino acids from dietary sugars. This, to our knowledge, is the first direct demonstration of amino acid transfer from pollen to butterfly eggs, and suggests that essential amino acids in particular are a key resource for extending lifespan and fecundity in Heliconius butterflies.     

Flower colour adaptation in a mimetic orchid

Although the tremendous variability in floral colour among angiosperms is often attributed to divergent selection by pollinators, it is usually difficult to preclude the possibility that floral colour shifts were driven by non-pollinator processes. Here, we examine the adaptive significance of flower colour in Disa ferruginea, a non-rewarding orchid that is thought to attract its butterfly pollinator by mimicking the flowers of sympatric nectar-producing species. Disa ferruginea has red flowers in the western part of its range and orange flowers in the eastern part--a colour shift that we hypothesized to be the outcome of selection for resemblance to different local nectar-producing plants. Using reciprocal translocations of red and orange phenotypes as well as arrays of artificial flowers, we found that the butterfly Aeropetes tulbaghia, the only pollinator of the orchid, preferred both the red phenotype and red artificial flowers in the west where its main nectar plant also has red flowers, and both the orange phenotype and orange artificial flowers in the east, where its main nectar plant has orange flowers. This phenotype by environment interaction demonstrates that the flower colour shift in D. ferruginea is adaptive and driven by local colour preference in its pollinator.     

Positive effects of cyanogenic glycosides in food plants on larval development of the common blue butterfly

Cyanogenesis is a widespread chemical defence mechanism in plants against herbivory. However, some specialised herbivores overcome this protection by different behavioural or metabolic mechanisms. In the present study, we investigated the effect of presence or absence of cyanogenic glycosides in birdsfoot trefoil (Lotus corniculatus, Fabaceae) on oviposition behaviour, larval preference, larval development, adult weight and nectar preference of the common blue butterfly (Polyommatus icarus, Lycaenidae). For oviposition behaviour there was a female-specific reaction to cyanogenic glycoside content; i.e. some females preferred to oviposit on cyanogenic over acyanogenic plants, while other females behaved in the opposite way. Freshly hatched larvae did not discriminate between the two plant morphs. Since the two plant morphs differed not only in their content of cyanogenic glycoside, but also in N and water content, we expected these differences to affect larval growth. Contrary to our expectations, larvae feeding on cyanogenic plants showed a faster development and stronger weight gain than larvae feeding on acyanogenic plants. Furthermore, female genotype affected development time, larval and pupal weight of the common blue butterfly. However, most effects detected in the larval phase disappeared for adult weight, indicating compensatory feeding of larvae. Adult butterflies reared on the two cyanogenic glycoside plant morphs did not differ in their nectar preference. But a gender-specific effect was found, where females preferred amino acid-rich nectar while males did not discriminate between the two nectar mimics. The presented results indicate that larvae of the common blue butterfly can metabolise the surplus of N in cyanogenic plants for growth. Additionally, the female-specific behaviour to oviposit preferably on cyanogenic or acyanogenic plant morphs and the female-genotype-specific responses in life history traits indicate the genetic flexibility of this butterfly species and its potential for local adaptation.     

Nutritional suitability of individual carbohydrates and amino acids for adult Pieris brassicae

Abstract Many adult Lepidoptera are dependent on carbohydrate-rich solutions such as nectar and honeydew. These food sources can contain a range of carbohydrates as well as low levels of other compounds, including free amino acids, proteins and lipids. Little is known about the nutritional value of individual nectar sugars and amino acids for adult Lepidoptera. Ten sugars occurring in nectar and honeydew were tested with respect to their effects on a number of Pieris brassicae life-table parameters. In similar experiments it was investigated whether the butterfly derives nutritional benefits from mixtures of common non-essential or essential amino acids when provided either in water or in a sucrose solution. Food sources were found to have a significant effect on butterfly fertility, longevity, lifetime fecundity and daily oviposition rate. Whereas a number of sugars significantly increase longevity, melibiose and melezitose reduce P. brassicae longevity. Butterfly fecundity is compromized significantly by sucrose and melibiose. Glucose is the only sugar that has a positive effect on both longevity and fecundity as well as a number of oviposition parameters. Amino acids seem to have no or only a minor effect on the life-table parameters tested. However, when provided in sucrose, they appear to compensate for the negative effect of pure sucrose on fecundity. A comparison with an earlier study on the gustatory response of P. brassicae shows that there is only a weak correlation between the nutritional value of sugars and amino acid mixtures and their perceptibility.     

Reversal Learning and Risk‐Averse Foraging Behavior in the Monarch Butterfly,Danaus plexippus (Lepidoptera: Nymphalidae)

Learning ability allows insects to respond to a variable environment, and to adjust their behaviors in response to positive or negative experiences. Pollinating insects readily learn to associate floral characteristics, such as color, shape, or pattern, with appetitive stimuli, such as the presence of a nectar reward. However, in nature pollinators may also encounter flowers that contain distasteful or toxic nectar, or offer highly variable nectar volumes, providing opportunities for aversive learning or risk‐averse foraging behavior. Whereas some bees learn to avoid flowers with unpalatable or unreliable nectar rewards, little is known about how Lepidoptera respond to such stimuli. We used a reversal learning paradigm to establish that monarch butterflies learn to discriminate against colored artificial flowers that contain salt solution, decreasing both number of probes and probing time on flowers of a preferred color and altogether avoiding artificial flowers of a non‐preferred color. In addition, when we offered butterflies artificial flowers of two different colors, both of which contained the same mean nectar volume but which differed in variance, the monarchs exhibited risk‐averse foraging: they probed the constant flowers significantly more than the variable ones, regardless of flower color or butterfly sex. Our results add to our understanding of butterfly foraging behavior, as they demonstrate that monarchs can respond to aversive as well as appetitive stimuli, and can also adjust their foraging behavior to avoid floral resources with high variance rewards.