Comparison of the Chemical Compositions of the Cuticle and Dufour's Gland of Two Solitary Bee Species from Laboratory and Field Conditions.

Species-specific biochemistry, morphology, and function of the Dufour's gland have been investigated for social bees and some non-social bee families. Most of the solitary bees previously examined are ground-nesting bees that use Dufour's gland secretions to line brood chambers. This study examines the chemistry of the cuticle and Dufour's gland of cavity-nesting Megachile rotundata and Osmia lignaria, which are species managed for crop pollination. Glandular and cuticular lipid compositions were characterized and compared to each other and according to the nesting experience of adult females. Major lipid classes found were hydrocarbons, free fatty acids, and wax esters. Many components were common to the cuticle and Dufour's glands of each species, yet not identical in number or relative composition. Wax esters and fatty acids were more prevalent in Dufour's glands of M. rotundata than on cuticles. Wax esters were more abundant on cuticles of O. lignaria than in Dufour's glands. In both species, fatty acids were more prevalent in glands of field-collected females compared to any other sample type. Chemical profiles of cuticles and glands were distinct from each other, and, for O. lignaria, profiles of laboratory-maintained bees could be distinguished from those of field-collected bees. Comparison of percentiles of individual components of cuticular and glandular profiles of the same bee showed that the proportions of some cuticular components were predictive of the proportion of the same glandular components, especially for nesting females. Lastly, evidence suggested that Dufour's gland is the major source of nest-marking substances in M. rotundata, but evidence for this role in O. lignaria was less conclusive.

Nest marking behavior and chemical composition of olfactory cues involved in nest recognition in Megachile rotundata.

In-nest observations of the solitary bee, Megachile rotundata (F.), revealed that nesting females apply olfactory cues to nests for nest recognition. On their way in and out of the nest, females drag the abdomen along the entire length of the nest, and sometimes deposit fluid droplets from the tip of the abdomen. The removal of bee-marked sections of the nest resulted in hesitation and searching behavior by females, indicating the loss of olfactory cues used for nest recognition. Chemical analysis of female cuticles and the deposits inside marked nesting tubes revealed the presence of hydrocarbons, wax esters, fatty aldehydes, and fatty alcohol acetate esters. Chemical compositions were similar across tube samples, but proportionally different from cuticular extracts. These findings reveal the importance of lipids as chemical signals for nest recognition and suggest that the nest-marking cues are derived from a source in addition to, or other than, the female cuticle.

The long summer: pre-wintering temperatures affect metabolic expenditure and winter survival in a solitary bee.

The impact of climate change on insect populations depends on specific life cycle traits and physiological adaptations. The solitary bee Osmia lignaria winters as a pre-emergent adult, and requires a period of cold temperature for winter diapause completion. It is a univoltine species, and diapause induction does not depend on photoperiod. To understand the potential effects of longer summers on O. lignaria populations, we exposed individuals to three treatments simulating early, mid and late winter arrivals, and measured respiration rates, metabolic expenditure, weight loss, fat body depletion, lipid levels and winter mortality. The early-winter treatment disrupted diapause development, but had no apparent negative effects on fitness. In contrast, late-winter bees had a greater energetic expenditure (1.5-fold), weight (1.4-fold) and lipid (2-fold) loss, greater fat body depletion, and a 19% increase in mortality compared to mid-winter bees. We also monitored adult eclosion and arrival of winter temperatures under natural conditions in four years. We found a positive correlation between mean degree-day accumulation during pre-wintering (a measure of asynchrony between adult eclosion and winter arrival) and yearly winter mortality. Individually, bees experiencing greater degree-day accumulations exhibited reduced post-winter longevity. Timing of adult eclosion in O. lignaria is dependent on the duration of the prepupal period, which occurs in mid-summer, is also diapause-mediated, and is longer in populations from southerly latitudes. In a global warming scenario, we expect long summer diapause phenotypes to replace short summer diapause phenotypes, effectively maintaining short pre-wintering periods in spite of delayed winter arrivals.

A comparison of internal and external lipids of nondiapausing and diapause initiation phase adult Colorado potato beetles, Leptinotarsa decemlineata.

The Colorado potato beetle, Leptinotarsa decemlineata, reared under diapause-inducing conditions will emerge from the soil as an adult and enter the diapause initiation phase, a period where metabolic reserves are stockpiled before the beetles enter the nonfeeding diapause maintenance phase. Internal and external lipids were characterized during the diapause initiation phase (IP) and compared to the lipid profiles of nondiapausing adults. The primary internal lipids of both diapause IP and nondiapausing adults are triacylglycerols. Only trace amounts of internal lipids were detected in day 1 diapause IP adults. A dramatic increase in internal lipids was observed between day 7 and day 15 post-emergence in the diapause IP adults. The majority of the triacylglycerol isomers were identified as C50, C52 and C54 chain lengths by GC-MS. There were no observed differences in the isomeric distribution of the major internal lipids between diapause IP and nondiapausing adults. External lipids were mainly methyl-branched alkanes containing a 25 to 53 carbon backbone. The quantity of external lipids increased from day 1 to day 7 post-emergence in both the diapause IP and nondiapausing adults, with the bulk of the increase occurring in the longer chain-length methylalkanes.

Internal lipids of sugarbeet root maggot (Tetanops myopaeformis) larvae: effects of multi-year cold storage.

Sugarbeet root maggots, Tetanops myopaeformis (Diptera, Ulidiidae), survive more than five years of laboratory cold (6 degrees C) storage as mature third-instar larvae. To quantify energy costs associated with prolonged storage, internal lipids of larvae stored for 1, 2, 3, and 5 years were compared and characterized with those of field-collected diapausing larvae. Internal lipid concentration was highest (21.8% wet wt. and 29.8% dry wt.) in diapausing larvae. Lipids decreased progressively over storage time with greater than 70% reductions for 5-year stored larvae. Thin-layer chromatographic analysis revealed that triacylglycerols (TAGs) were the most predominant class of internal lipids, with trace amounts of diacylglycerols and hydrocarbons also being present. Gas chromatography-mass spectrometry (GC-MS) analyses of TAG fractions identified ten major fatty acids (FAs). The proportion of unsaturated FAs was higher (73 to 78%) than saturated FAs in diapausing and stored larval groups. Palmitoleic acid (16:1) was the predominant FA, constituting 40-50% of total unsaturated FAs with lesser amounts of myristoleic (14:1), oleic (18:1), lauroleic (12:1), gadoleic (20:1), and the saturated FAs, palmitic (16:0), myristic (14:0), lauric (12:0), stearic (18:0), and arachidic (20:0) being detected at much lower concentrations. Characterization of intact TAGs by high performance liquid chromatography and GC-MS revealed the presence of more than 40 TAG constituents. In conclusion, TAGs are utilized as an important energy source for T. myopaeformis larvae during diapause and long-term cold storage with no observed impact of multi-year storage on the TAG composition and distribution of their fatty acids.

Cuticular lipids of female solitary bees, Osmia lignaria Say and Megachile rotundata (F.) (Hymenoptera: Megachilidae).

The cuticular lipids of the cavity-nesting adult female solitary bees, Osmia lignaria Say and Megachile rotundata (F.) (Hymenoptera: Megachilidae), were analyzed by gas chromatography (GC) and combined GC-mass spectrometry. The cuticular lipids of these female bees are mainly consisted of hydrocarbons. For O. lignaria, nearly 64% of the cuticular lipids were C(25)-C(31) mono-alkenes. For M. rotundata, 48% of the cuticular lipids were C(23)-C(33) alkanes with nearly the same quantities of the same chain-length mono-alkenes (45%). For the mono-alkenes of O. lignaria, 14 mono-alkene constituents were identified, with two of these, 9-heptacosene and 7-nonacosene, comprising 67% of the total alkene distribution. For M. rotundata females, the mixtures of mono-alkenes were more complex with 26 constituents identified and quantified. For the M. rotundata mono-alkenes, 57% of the total composition consisted of the three alkenes, 7-pentacosene, 9-pentacosene and 7-heptacosene. For both bee species, small quantities of C(40)-C(48) wax esters were also characterized with the major components possessing a C(18) mono-unsaturated fatty acid (9-octadecenoate) moiety esterified to even-carbon number (C(22-30)) fatty alcohols. The possible role of these cuticular lipids as nest recognition chemicals is discussed in light of nesting behavior of managed crop pollinators.

Transfer of methyl-branched hydrocarbons from the parasitoid, Eretmocerus mundus, to silverleaf whitefly nymphs during oviposition.

The parasitic wasp Eretmocerus mundus (Hymenoptera: Aphelinidae), a natural enemy of the silverleaf whitefly Bemisia argentifolii (Homoptera: Aleyrodidae), deposits eggs beneath nymphs and not within them. Experiments were designed to establish whether ovipositing E. mundus females leave marking chemicals on nymphs to enable searching females to discriminate parasitized from unparasitized hosts. Cuticular lipids from three experimental treatment groups were characterized: parasitoid-exposed nymphs that had a parasitoid egg between the nymph and leaf; control nymphs not exposed to E. mundus; and a third treatment condition of parasitized nymphs, held for 10 days after wasp exposure. Lipids were solvent-extracted from the nymphal cuticles of the various treatment groups and the lipid components were characterized and quantified by gas chromatography and mass spectrometry. Results indicated the presence of quantities of C31 and C33 dimethylalkanes only from parasitoid-exposed groups of nymphs and not in the extracts from control nymphs or the parasitized nymphs after 10-day exposure. Furthermore, the C31 and C33 dimethylalkanes were shown to be major lipid components of the hexane extracts from E. mundus females. Since the lipids were removed from parasitoid-exposed nymphs before interaction with hatched parasitoid larvae, the findings indicated that the dimethylalkanes were transferred onto nymphal cuticles by ovipositing E. mundus females.

Characterization of triacylglycerols from overwintering prepupae of the alfalfa pollinator Megachile rotundata (Hymenoptera: Megachilidae).

Alfalfa leafcutting bees, Megachile rotundata (F.), overwinter as prepupae. The internal lipids were extracted from prepupae that had been wintered at 4 degrees C for 7 months. Megachile rotundata prepupae possessed copious quantities of internal lipids (20% of the fresh weight) that were extracted with CHCl3/methanol (2:1). Transmission electron microscopy revealed that lipids were stored within very large intracellular vacuoles. Separation by silica chromatography revealed that 88% of the internal lipids were triacylglycerols. Ester derivatives of fatty acids from triacylglycerol components were analyzed by gas chromatography-mass spectrometry and 15 fatty acid constituents were identified. The majority (76%) of the triacylglycerol fatty acids were unsaturated fatty acids. The major triacylglycerol fatty acid constituent (30%) was the C16 monounsaturated fatty acid, palmitoleic acid (16:1, hexadec-9-enoic acid), with substantial amounts of linolenic acid (18:3, octadec-9,12,15-trienoic acid, 15%), palmitic acid (16:0, hexadecanoic acid, 14%) and oleic acid (18:1, octadec-9-enoic acid, 13%). Palmitoleic acid as the major fatty acid of an insect is an unusual occurrence as well as the presence of the 16-carbon polyunsaturated fatty acids, 16:2 and 16:3. The major intact triacylglycerol components were separated and identified by high performance liquid chromatography-mass spectrometry. A complex mixture of approximately 40 triacylglycerol components were identified and major components included palmitoyl palmitoleoyl oleoyl glycerol, palmitoyl palmitoleoyl palmitoleoyl glycerol, myristoyl palmitoleoyl palmitoleoyl glycerol, myristoleoyl palmitoyl palmitoleoyl glycerol, and palmitoyl palmitoleoyl linolenoyl glycerol. The function of these internal lipids and their relevance to winter survival and post-wintering development of M. rotundata is discussed.

Characterization of the cuticular surface wax pores and the waxy particles of the dustywing, Semidalis flinti (Neuroptera: Coniopterygidae).

The adult dustywing, Semidalis flinti Meinander (Neuroptera: Coniopterygidae), begins producing circular-shaped waxy particles after eclosion. The waxy material, which forms the particles, is extruded from individual pores found in clusters on the abdomen. Pores also are present in two rows of three pores on the frontalis and two pores on the first segment of each antennae. The pores have a rosette-like appearance and each pore extrudes dual waxy ribbons. As each ribbon extends a short distance out of the pore, it begins to curl back on itself until the end makes contact with the ribbon. The curled end then breaks free from the extruding ribbon to form the circular waxy particles with fluted edges approximately 2.75-microm diameter. The adults use the particles to cover all parts of their body except for their eyes and appear to lightly coat their antennae. The lipid portion of the particles consists largely of free fatty acids, almost exclusively the 24-carbon fatty acid, tetracosanoic acid. Minor lipid classes are hydrocarbons, fatty alcohols and unidentified material.

Triacylglycerol and phospholipid fatty acids of the silverleaf whitefly: composition and biosynthesis.

The identification and composition of the fatty acids of the major lipid classes (triacylglycerols and phospholipids) within Bemisia argentifolii Bellows and Perring (Homoptera: Aleyrodidae) nymphs were determined. Comparisons were made to fatty acids from the internal lipids of B. argentifolii adults. The fatty acids, as ester derivatives, were analyzed by capillary gas chromatography (CGC) and CGC-mass spectrometry (MS). All lipid classes contained variable distributions of eight fatty acids: the saturated fatty acids, myristic acid (14:0), palmitic acid (16:0), stearic acid (18:0), arachidic acid (20:0); the monounsaturated fatty acids, palmitoleic acid (16:1), oleic acid (18:1); the polyunsaturated fatty acids, linoleic acid (18:2), linolenic acid (18:3). Fourth instar nymphs had 5-10 times the quantities of fatty acids as compared to third instar nymphs and 1-3 times the quantities from adults. The fatty acid quantity differences between fourth and third instar nymphs were related to their size and weight differences. The percentage compositions for fatty acids from each lipid class were the same for the pooled groups of third and fourth instar nymphs. For nymphs and adults, triacylglycerols were the major source of fatty acids, with 18:1 and 16:0 acids as major components and the majority of the polyunsaturated fatty acids, 18:2 and 18:3 were present in the two phospholipid fractions, phosphatidylethanolamine and phosphatidylcholine. Evidence was obtained that whiteflies indeed synthesize linoleic acid and linolenic acid de novo: radiolabel from [2-(14)C] acetate was incorporated into 18:2 and 18:3 fatty acids of B. argentifolii adults and CGC-MS of pyrrolidide derivatives established double bonds in the Delta(9,12) and Delta(9,12,15) positions, respectively.

Mouthpart morphology and stylet penetration of host plants by the glassy-winged sharpshooter, Homalodisca coagulata, (Homoptera: Cicadellidae).

The ultrastructural morphology of the mouthparts of the glassy-winged sharpshooter, Homalodisca coagulata, and method of plant penetration was examined using light microscopy, scanning electron microscopy, and transmission electron microscopy methods. The gross morphology of the labrum, labium, and stylet fascicle was consistent with what has been described for other plant-sucking homopterans. The ultrastructural examination of the mouthparts revealed unique details that have previously gone unreported. Several types of sensilla-like structures having the form of pegs and multi-lobed objects were identified on the outer surfaces of the labrum and within the labial groove. Dendritic canals terminated in an extensive network of smaller canals at the distal tip of the maxillary stylets below a series of surface denticles suggesting that this area may have a sensory function associated with locating xylem elements of host plants. Examination of salivary sheath pathways established that 65% of the plant penetrations by this insect terminated in the xylem vessels of the host plant. Probing by the insect was largely intracellular and terminal branching of a single probe site was common. Plant surface feeding sites varied with the stage of development which correlates with the depth of the xylem vessels and the length of the maxillary stylets of the various instars.

Characterization and functions of the whitefly egg pedicel.

For the silverleaf whitefly, Bemisia argentifolii (Bellows and Perring) (Homoptera: Aleyrodidae), scanning and transmission electron microscopic techniques were used to observe the characteristics of egg oviposition into both plant cells/tissues and artificial membranes, and to document the morphology of mature egg pedicles removed from the ovaries of females. The exterior of the distal portion of the pedicel consisted of a tangled array of fibrous structures (0.2-0.3 microm in diameter) that constituted about 20-25% of the outer diameter of the pedicel. The attachments of the fibers to the core of the pedicle suggested that the pedicel functions as the collector and conduit for water (vapor), and perhaps solute movement into the egg. Silverleaf whitefly eggs on membranes were incubated at various levels of relative humidity and the eggs were scored for egg hatch. At 98-100% rh, the percentage egg hatch was 86-98%. At lower humidity ranges of 0-20, 55-65, and 75-85% rh, none of the eggs hatched. Media (solute) uptake by silverleaf whitefly egg pedicels was determined by exposing the pedicel side of eggs oviposited on membranes to media solutions containing the high molecular weight polysaccharide, [(14)C]-inulin. Solute uptake by the pedicel and movement into developing silverleaf whitefly eggs were demonstrated using [2-(14)C]-acetate, and assaying for radioactivity in hatched nymphs. These studies, using exposure of pedicels to relative humidity and radiolabeled materials, demonstrate that whitefly egg hatch is dependent upon water uptake by the pedicel, and that the pedicel has the ability to transport solutes into the developing egg.