Naturwissenschaften (2012) 99:695–703 DOI 10.1007/s00114-012-0951-z
ORIGINAL PAPER
Procurement of exogenous ammonia by the swallowtail butterfly, Papilio polytes, for protein biosynthesis and sperm production Keiichi Honda & Hiroyuki Takase & Hisashi Ômura & Hiroshi Honda Received: 13 March 2012 / Revised: 4 July 2012 / Accepted: 16 July 2012 / Published online: 28 July 2012 # Springer-Verlag 2012
Abstract How to acquire sufficient quantity of nitrogen is a pivotal issue for herbivores, particularly for lepidopterans (butterflies and moths) of which diet quality greatly differs among their life stages. Male Lepidoptera often feed from mud puddles, dung, and carrion, a behavior known as puddling, which is thought to be supplementary feeding targeted chiefly at sodium. During copulation, males transfer a spermatophore to females that contains, besides sperm, nutrients (nuptial gifts) rich in sodium, proteins, and amino acids. However, it is still poorly understood how adults, mostly nectarivores, extract nitrogen from the environment. We examined the availability of two ubiquitous inorganic nitrogenous ions in nature, viz. ammonium (or ammonia) and nitrate ions, as nutrients in a butterfly, and show that exogenous ammonia ingested by adult males of the swallowtail, Papilio polytes, can serve as a resource for protein biosynthesis. Feeding experiments with 15N-labeled ammonium chloride revealed that nitrogen was incorporated into eupyrene spermatozoa, seminal protein, and thoracic muscle. Ammonia uptake by males significantly increased the number of eupyrene sperms in the reproductive tract tissues. The females also had the capacity to assimilate ammonia into egg protein. Consequently, it is evident that acquired ammonia is utilized for the replenishment of proteins allocable for reproduction and somatic maintenance. The active exploitation of exogenous ammonia as a nutrient by a butterfly
Communicated by: Sven Thatje K. Honda (*) : H. Takase : H. Ômura Department of Biofunctional Science and Technology, Graduate School of Biosphere Science, Hiroshima University, Higashihiroshima 739-8528, Japan e-mail:
[email protected] H. Honda Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba 305-8572, Japan
would foster better understanding of the foraging and reproductive strategies in insects. Keywords Puddling behavior . Nutrition . Nitrogen . Reproduction . Sperm competition
Introduction Nutrition is a common issue of vital importance for all organisms, particularly for heterotrophs that depend exclusively on reduced carbon compounds produced by plants to sustain their lives. Among a variety of nutrients animals require, nitrogenous substances, such as proteins and amino acids, are the ingredients indispensable for their growth, development, and reproduction. Herbivores generally live on low-nitrogen diets as compared to carnivores, and in herbivorous insects (about half of the insects), their reproductive success is often restrained due to the shortage of dietary nitrogen. For adults of herbivorous lepidopterans, how to allocate their nutrient budgets to reproductive output is a more crucial issue because diet quality and energy demands greatly differ among life stages (Boggs 1981a; Braby and Jones 1995). In particular, the availability of nitrogen, an element of utmost importance for reproduction (Marshall 1982; Bissoondath and Wiklund 1995), is limited as they feed on nitrogen-deficient diets, such as floral nectar, rotting fruits, or exuded tree sap, though some heliconiine butterflies can feed on pollen (Gilbert 1972), which is rich in amino acids and proteins. It has been generally believed that nutrients acquired as larvae and carried over to adults are the major storage reserves they can expend for reproduction and somatic maintenance (Boggs 1981a; Telang et al. 2001; O’Brien et al. 2002). However, recent studies have shown that female income as an adult also contributes to its fitness parameters (e.g., fecundity and egg hatching success) under certain circumstances (Mevi-Schütz and Erhardt 2005;
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Geister et al. 2008). Some tropical male butterflies also benefit from amino acid feeding in attaining their long life spans (Beck 2007). Adults of some charaxine butterflies (Nymphalidae) that are capable of fast and powerful flight often feed on carrion, presumably in order to meet additional nitrogen requirements resulting from greater musculature (Hamer et al. 2006). These suggest that acquisition of nitrogen as an adult is no less important than larval feeding. In Lepidoptera, males transfer two vastly different types of sperms, the eupyrene (nucleate) and apyrene (anucleate) spermatozoa, to females via the spermatophore at mating (Friedländer et al. 2005). Males also provide females with nutritious accessory gland products, which are absorbed by the female and used to manufacture eggs (Boggs and Gilbert 1979). The increment in female’s resource budget due to paternal investment adds to female fitness; in some species, a positive relationship is found between the amount of male ejaculate received and lifetime fecundity (Oberhauser 1997; Karlsson 1998). The non-sperm portion of seminal fluid substances consists of a diverse array of nutrients including proteins, amino acids, saccharides, phospholipids, and hydrocarbons (Leopold 1976; Marshall 1985; Bissoondath and Wiklund 1995), and evidence suggests that males, as well as females engaging in egg production, may incur considerable physiological cost when producing a spermatophore (Boggs 1981b; Svärd and Wiklund 1986). Puddling, most frequently seen in Lepidoptera, is a behavior in which adults feed from mud, dung, and carrion, occasionally in large groups (Norris 1936; Downes 1973). This behavior is heavily male-biased in most species, and is thought to be supplementary feeding aimed to acquire sodium, because (1) male Lepidoptera donate, in addition to various nutrients, substantial amounts of sodium to females at mating (Adler and Pearson 1982; Pivnick and McNeil 1987; Smedley and Eisner 1996), (2) sodium, usually a scarce element in plants (Wallace et al. 1948), is not readily available to lepidopterans (mostly folivores and nectarivores), and (3) they are stimulated to feed by sodium, strongly prefer sodium-supplemented mud puddles, and actually ingest it (Arms et al. 1974; Smedley and Eisner 1995; Boggs and Dau 2004). In this sense, puddling in insects is somewhat similar to geophagy (soil ingestion), a phenomenon widespread in many vertebrates such as elephants, primates, and birds that acquire minerals from soil (e.g., Hold et al. 2002; Mahaney and Krishnamani 2003). Since carrion, for example, can be a source of proteins and amino acids, it has been postulated that nitrogenous substances are also sought by males. In fact, preferences for some proteins, amino acids, or ammonium ion have also been demonstrated in the context of puddling (Arms et al. 1974; Beck et al. 1999) and nectar feeding (Erhardt and Rusterholz 1998), though female preference for amino acids seems to depend on her history of larval feeding and mating (Mevi-Schütz
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and Erhardt 2003, 2004). However, it remains unclear whether males actually intake nitrogenous compounds through puddling (Molleman and Midgley 2009; Molleman 2010). Furthermore, the effect of male-acquired sodium and/ or nitrogenous substances on its own reproductive ability or neuromuscular activity (Lederhouse et al. 1990; Hall and Willmott 2000; Watanabe and Kamikubo 2005; Molleman et al. 2005; Lewis and Wedell 2007) and on female reproductive output (Pivnick and McNeil 1987; Lederhouse et al. 1990; Molleman et al. 2004) is disputed. Thus, very little is known about what benefit accrues to males from puddling. The point worthy of investigation in regard to nutritional ecology is to uncover the strategy by which nectar-feeding insects procure nitrogen to maximize their reproductive capacity. Here we examined whether the tropical swallowtail butterfly Papilio polytes, one of typical puddlers, can utilize nitrogen-containing inorganic ions that are ubiquitous in nature, viz. ammonium (cation) and nitrate (anion) ions (Feth 1966), as nutrients, by means of stable isotope (15N) experiments where necessary. The questions addressed in this study are as follows: (1) which of some naturally occurring ions do males of the butterfly preferentially take in?; (2) whether the males can assimilate the two nitrogenous ions into proteins (amino acids); (3) if so, whether both sexes can exploit ammonia for protein production; and (4) how ammonia ingestion will affect male reproductive potential (changes in sperm counts and the amount of seminal protein, for example).
Materials and methods Insects Adult males of P. polytes used for the experiments were progeny of females collected in the Yaeyama Islands, Okinawa prefecture, Japan. Larvae were reared at 25 °C under a 16L/8D regime on potted host plants of the butterfly, Citrus spp. and Glycosmis citrifolia (Rutaceae). Adults used in the experiments were all from non-diapause pupae, unless otherwise noted. In all experiments except ion absorption tests, the difference in mean forewing length of males between control and treated cohorts in each set of experiment was below 1 mm. Chemicals 15
N-labeled ammonium chloride (15NH4Cl; 99 at.%) and sodium nitrate (Na15NO3; 99 at.%) were purchased from MSD Isotopes. Sodium chloride, sodium nitrate, dipotassium hydrogenphosphate, and ammonium sulfate were all reagent grade (Nacalai tesque).
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Absorption rate of ions This experiment was carried out to examine which ion(s) are preferentially absorbed by P. polytes males. A puddling male imbibes water uninterruptedly for minutes, during which it voids a fluid at intervals of seconds. Three- to eight-day-old males were subjected to the experiment. A male adult placed in a transparent plastic chamber (33× 42 cm; height, 24 cm) kept at 25 °C was allowed to drink salt water ad libitum containing equi-molar [either 0.025 M (A) or 0.0125 M (B) each] sodium chloride, sodium nitrate, dipotassium hydrogenphosphate, and ammonium sulfate. The total concentration of salts in solution A is 0.1 M and its ion concentration is 0.05 M for each cation, and 0.025 M for each anion. In this experiment, summer males (N09) that had emerged from non-diapause pupae and spring males (N09) from overwintering (diapausing) pupae were used. Based on preliminary preference tests, summer males were fed solution A or B, while spring males, solution B. After discarding the first two to three drops of the excretion, ejected fluids were collected for minutes. Net absorption of cations and anions was calculated from the differences in ion concentrations between the imbibed and ejected fluids, employing an ICS-1600 ion chromatograph (DIONEX). Cations were analyzed on IONPAC CS212A (4 mm i.d.× 250 mm; DIONEX), using 20 mM aq. methanesulfonic acid as an eluent, and anions, on IONPAC AS22 (4 mm i.d.× 250 mm; DIONEX), using an aq. carbonate solution consisting of sodium hydrogen carbonate (1.0 mM) and sodium carbonate (4.5 mM) as an eluent. The flow rate was 1.0 ml for cations and 1.2 ml for anions. The column effluent was monitored with a conductivity detector embedded in the IC system. Oral administration of salts and tissue sample collection From the day following eclosion, individual males were fed daily with 50 μl of any one of four 50 mM salt solutions dissolved in 20 % sucrose for five successive days, by applying droplets of the solution to the coiled proboscis with a microsyringe. The salts tested were 15 NH 4 Cl, Na15NO3, NH4Cl, and NaCl. Control males were fed 20 % plain sucrose only. Males were kept in a transparent plastic chamber (33×42 cm; height, 24 cm) at 25 °C under a 16L/ 8D regime (ca. 500 lx) throughout the experiment. The chamber was illuminated (ca. 3,500 lx) for at least 2 h every day to permit them free flight. On post-emergence day 7, reproductive tract tissues and mesothoracic musculature were excised in Ringer’s solution. From the day following eclosion, females (N05) were fed daily with 50 μl of 50 mM 15NH4Cl solution for five successive days and kept at 25 °C in the same manner as males. Eggs were obtained from 7- to 9-day-old females by allowing them
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to oviposit on Citrus plants as hosts. The first 30 eggs laid after the onset of oviposition were collected for 15N analysis. Isotope (15N) measurement and sample preparation The abundance (atomic percent) of 15N in test materials was measured with a DELTAplus Advantage IRMS system after combusting samples in a Flash EA1112 combustion furnace (Thermo Fisher Scientific). The proportion of 15N was determined for crude organic matters (COM; mainly protein and sperm) in the male reproductive tract tissues (testis, accessory gland, vas deferens, seminal vesicle, and ejaculatory duct), eupyrene sperm, seminal fluid protein, male mesothoracic musculature, and unfertilized eggs deposited. For the preparation of COM (N08 for 15NH4Cl, N03 for Na15NO3, and N03 for control), all tissues in the male reproductive tract were carefully excised in Ringer’s solution and gently rinsed with distilled water three times. The tissues were macerated in 1 ml of distilled water and dissected with forceps. After having taken out the tissues completely, 2 ml of ethanol was added to the liquor with stirring. The whole liquor was centrifuged at 2,500×g for 10 min at room temperature (RT) and the supernatant was discarded to remove inorganic materials. The precipitate was washed with 70 % aq. ethanol, dried at 60 °C, and pulverized for 15N analysis. The eupyrene sperm bundles were collected from the testis. The testis was dissected in distilled water and the content was centrifuged at 2,500×g for 10 min at RT. The supernatant was discarded and the precipitate was washed successively with water and 70 % aq. ethanol by centrifugation to remove remaining apyrene sperms and lipophilic substances, and dried at 60 °C. To fractionate seminal protein, all tissues in the male reproductive tract were dissected in distilled water and the content was centrifuged at 2,500×g for 10 min at RT. The supernatant fluid was filtered three times with a membrane filter (0.22 μm, MS MCE Syringe Filter) and to the filtrate was added a large amount of ethanol to adjust its concentration at ca. 70 %. After heating the entire liquor at 80 °C for 1 h to denature protein, the liquor was centrifuged at 2,500×g for 10 min at RT. The precipitate was washed with 70 % aq. ethanol and dried at 60 °C. Due to the paucity of eupyrene spermatozoa and seminal fluid proteins collected, each sample (N05) of these for 15N analysis consisted of pooled materials prepared from four to seven individuals. The muscular tissue excised from the male mesothorax (N05) was washed with distilled water and dried at 60 °C. Eggs deposited were detached from leaves, washed with distilled water twice to remove surface substances, and crushed in 70 % aq. ethanol. After having taken out the egg shell, the liquor was centrifuged at 2,500×g for 10 min at RT. The supernatant was discarded and the precipitate was washed again with 70 % aq. ethanol and dried at 60 °C.
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Amino acid analysis
Gross nitrogen content of reproductive tissue products
The COM prepared from males fed with 15NH4Cl (N05) or Na15NO3 (N05) as described above was hydrolyzed with 18 % aq. HCl at 100 °C for 24 h. The amino acid composition of the hydrolysate (water-soluble substances) was determined with a JEOL JLC-500/V2 amino acid analyzer.
Nitrogen content of gross products in the male reproductive tract tissues prepared as described above (N014 for NH4Cl, N011 for NaCl, and N09 for control) was measured with a PerkinElmer CHNS/O 2400II elemental analyzer. Statistics
Sperm counts The number of eupyrene and apyrene sperms in the male reproductive tract tissues was examined for males fed with unlabeled NH4Cl or NaCl and for control males (N010 for each sample). All reproductive tissues excised were dissected with forceps in Ringer’s solution, which was subsequently diluted up to 4 ml (A) with gentle stirring. An aliquot (50 μl) of liquor A was dropped on a hole glass slide and the number of eupyrene sperm bundles was counted with a phase-contrast microscope (magnification ×40). The measurement was replicated five times and the values averaged. The total eupyrene sperm count was calculated by multiplying the mean by 256 (Virkki 1969) and the dilution factor. For counting the number of apyrene sperm, liquor A was further subjected to 1,500- to 2,500-fold dilution with Ringer’s solution. The number of apyrene sperm in a 4-μl portion of the diluted liquor was counted in a similar manner (×100). The measurement was replicated five times and the values averaged. The total apyrene sperm count was calculated by multiplying the mean by the dilution factor. Quantification of seminal protein The quantity of seminal protein was measured using a dyebinding protein assay according to the standard method (Bradford 1976; Avila et al. 2011). Male reproductive tract tissues (N010 each for NH4Cl, NaCl, and control) were dissected in Ringer’s solution and the entire content was filtered with a membrane filter (0.22 μm). An aliquot of the filtrate was mixed with a phosphate buffer solution (pH 6.8) and a dye (Coomassie Brilliant Blue G-250) standard solution. The mixture was left standing for more than 5 min at RT and subjected to color photometry within 1 h of sample preparation. The photometric analysis was carried out with a Shimadzu UV mini 1240 spectrophotometer by measuring the absorption at 595 nm. The protein content was estimated on the basis of a bovine serum albumin calibration curve. Dry mass of reproductive tissue products All tissues in the male reproductive tract (N017 for NH4Cl, N011 for NaCl, and N010 for control) were macerated and dissected in distilled water, after which the entire content was lyophilized and weighed.
Significance of difference in incorporation rate of 15N into COM in male reproductive tract tissues was tested by an Aspin–Welch’s t test, while one-way ANOVA was used for analyzing the influences of ammonia or sodium uptake on male reproductive parameters (amount of seminal protein, dry mass of reproductive tissue products, and gross nitrogen content of reproductive tissue products). In the other experiments except ion absorption tests, significance of difference between control and treatments or between treatments was assessed by a Mann–Whitney U test.
Results Absorption rate of ions We measured the absorption rate of three cations, sodium, potassium, and ammonium, and four anions, chloride, nitrate, phosphate, and sulfate. Summer males showed a relatively high absorption rate, roughly 40–70 % for cations and 50–70 % for anions, irrespective of the concentration of salt solution imbibed (Table 1). In contrast, spring males tended to less actively absorb ions; 20–30 % for cations and 20– 40 % for anions. Apart from seasonal variation of the absorption rate, its individual variation was very large in both summer and spring males. Consequently, no conclusive answer was obtained as to which ion was preferentially absorbed. Based on the normalized values, however, it appears that ammonium ion might be absorbed somewhat efficiently by summer males that are known to frequent mud puddles, while potassium and chloride ions might be more positively absorbed by spring males. Incorporation of tissues
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N into COM in male reproductive tract
This experiment tested whether ammonium and/or nitrate ions were utilized for protein biosynthesis. The COM present in the reproductive tract tissues of males fed 15N-labeled ammonium chloride (15NH 4Cl) showed a significantly higher percentage (mean ± SD; 2.00±0.48 %) of 15N than those derived from control males (0.372±0.001 %) (t0 8.989, df07.000, p
