Storage proteins of the fall webworm,Hyphantria cunea drury

Two storage proteins, storage protein-1 (SP1) and storage protein-2 (SP2), were found in hemolymph and fat body during the development of Hyphantria cunea, the fall webworm. Both storage proteins show similiar quantitative changes during development in males and females; however, SP1 is more abundant. The hemolymph of last instar larvae contains high concentrations of the storage proteins. However, following pupation, the storage proteins accumulate in fat bodies. SP1 peaks in the hemolymph of males and females late in last instar larvae (8-day-old 7th instar larvae). SP1 has a native molecular weight of 460,000 and consists of six identical subunits (Mr = 76,700), while SP2 has a molecular weight of 450,000 and is composed of two different subunits (Mr = 74,100 and 72,400). Both SP1 and SP2 are hexamers and are phosphorylated glycolipoproteins. The pl values of SP1 and SP2 were determined to be 5.70 and 5.50, respectively. Antibodies raised against SP1 react positively with vitellogenin and ovary extract, as well as with proteins in the hemolymph from last instar larvae and proteins in pupal fat bodies. Storage protein synthesis starts in fat bodies of a 4-day-old 7th instar larvae and in female peaks at 6–8 days of the 7th instar.     

Purification,characterization, and synthesis of vitellin from the cabbage butterfly Pieris rapae L.

Vitellin from the cabbage butterfly Pieris rapae L. was purified and characterized by electrophoresis. Vitellin from P. rapae is a phosphorylated glycolipoprotein of 380,000 ± 10,000 molecular weight as determined by nondenaturing polyacrylamide gel electrophoresis. Two subunits with an M_r of 150,000 and 40,000 were obtained from vitellin. The native molecule is thought to be a tetramer composed of two molecules of each of these subunits. The isoelectric point, as determined by isoelectric focusing on polyacrylamide gels, is 6.10. Vitellin and vitellogenin were indistinguishable by immunological methods such as double diffusion and tandem-crossed immunoelectrophoresis. Vitellogenin from the hemolymph and vitellin from the ovary were quantified by rocket immunoelectrophoresis. Vitellogenin and vitellin were first detected in 6-day-old pupae, and their levels increased continuously during ovarian development. Vitellogenin synthesis by the fat body in 4-day-old female pupae could be induced by juvenile hormone I.     

Ovariectomy in grasshoppers increases somatic storage, but proportional allocation of ingested nutrients to somatic tissues is unchanged

Reduced reproduction increases storage and extends lifespan in several animal species. The disposable soma hypothesis suggests this life extension occurs by shifting allocation of ingested nutrients from reproduction to the soma. A great deal of circumstantial evidence supports this hypothesis, but no direct tracking of nutrients has been performed in animals that are long-lived because of direct reduction in reproduction. Here, we use the stable isotopes to track carbon and nitrogen from ingestion to somatic organs in long-lived, ovariectomized grasshoppers. Three estimates of somatic storage (viz., quantity of hemolymph storage proteins, amount of femur muscle carbohydrates, and size of the fat body) all doubled upon ovariectomy. In stark contrast, ovariectomy did not increase the proportion of these tissues that were made from recently ingested foods. In other words, the physiology underlying relative allocation to these somatic tissues was not affected by ovariectomy. Thus, at the level of whole tissue storage, these results are consistent with a trade-off between reproduction and longevity. In contrast, our stable isotope data are inconsistent with the prediction that enhanced storage in ovariectomized females results from a physiological shift in allocation of ingested nutrients.     

The effect of host acceptability on oviposition and egg accumulation by the small white butterfly, Pieris rapae

The influence of host‐plant acceptability on oviposition rate, egg load, internal fat storage and longevity was studied in the small white butterfly, Pieris rapae L. (Lepidoptera: Pieridae). Newly emerged females and males were presented with either cabbage (Brassica oleracea L. var. gemmifera), which is a highly acceptable host for the small white, or wallflower (Cheiranthus cheiri L.), which is much less acceptable for oviposition. Individuals were dissected when 3, 6 and 9‐days‐old. Females kept on cabbage contained fewer mature eggs than those kept on wallflower and there was no change in the load of mature eggs over the time course studied. The number of immature eggs carried did not vary with host plant species, but did fall significantly between days 6 and 9. The body fat content of individuals declined with increasing age, but the decline was slower for individuals kept on the host of low acceptability. Individuals that were allowed to spend their natural lifespan on cabbage showed similar oviposition patterns over time, where the oviposition rate started high and gradually reduced until death. However, considerable differences in oviposition pattern were found in individuals kept on wallflower, varying from that found on cabbage to no oviposition at all. The implications of these findings are discussed in the light of existing oviposition theories. This leads to the conclusion that in one species the premises of existing theories on optimization of oviposition are not mutually exclusive but rather play in concert.     

Vitellogenesis and oocyte development in azadirachtin-induced fifth-instar overage nymphs of Locusta migratoria (L.)

Injection of azadirachtin into females of Locusta migratoria at the beginning of the last nymphal instar prevented molting to the adult stage, and many of these locusts survived for long periods as overage fifth-instar nymphs. Overage female nymphs synthesized vitellogenin; maximum vitellogenin content in their hemolymph was 6–7 times higher than that found in normal adult females. The overage female nymphs developed vitellogenic oocytes, but development was retarded to some extent: although vitellogenin did accumulate in the proximal oocytes, their maximum average length was only about 2.8 mm (compared to 6.2 mm in normal adult females) and extensive oocyte resorption was observed. Thus, attainment of adult competence of the organs and processes involved in female reproduction is independent to a considerable extent from the process of overt adult morphogenesis.     

Effect of phase status on responses to AKHI in the desert locust,Schistocerca gregaria gregaria

Hyperlipaemic response to adipokinetic hormone (AKH I) was demonstrated in both solitary and gregarious phases of the desert locust, Schistocerca gregaria gregaria. Time-course studies showed that the gregarious locusts had a faster response to the hormone than their solitary counterparts. At peak response time (90 min), the gregarious locusts were more sensitive to AKH I doses below 2 pmol while the solitary locusts had a higher response above this dose. Upon injection of the hormone, lipoprotein conversion occurred, resulting in the formation of the low density lipoprotein (LDLp). The LDLp formed in the gregarious locusts was much larger than that of the solitary locusts. The fat body lipid reserve (expressed as % fat body dry weight) was significantly (P < 0.01) higher in the gregarious (79.02 ± 2.77%) than in the solitary locusts (65.23 ± 2.55%). Triacylglycerol was the major lipid class representing 83.9 and 73.9% of the total lipids in gregarious and solitary locusts, respectively. The higher fat body lipid reserves and efficient LDLp formation in response to AKH in gregarious locusts compared to solitary locusts suggests a physiological adaptation for prolonged flights. © 1996 Wiley-Liss, Inc.     

Occurrence and accumulation of the granulosis virus of Pieris rapae in treated field plots

The granulosis virus of Pieris rapae, the imported cabbageworm (P. rapae GV), persisted in soil after application. Virus produced by epizootics of the disease in populations of the host accumulated in soil in nontreated plots with concentrations of the virus in soil and on foliage, being similar at harvest to concentrations in virus-treated plots. Also P. rapae GV produced in epizootics of the disease increased concentrations in soil of virus-treated plots and maintained substantial residues of the virus on the foliage. Little virus accumulated in plots treated with some chemical insecticides and Bacillus thuringiensis because few host larvae survived to support late-season epizootics. Small quantities of P. rapae GV were found in heads of cabbage harvested from plots in October, but these residues were not related to plot treatment.A study in which nontreated and virus-treated plots were replanted for 2 years after treatment indicated an increase in concentrations of P. rapae GV in surface soil in years following treatment. Concentrations of P. rapae GV in soil in virus-treated and nontreated plots were similar in the autumn of the year of treatment and in subsequent years of the study. Concentrations were nearly unchanged during the winter, were reduced by cultivation preparatory to planting in the spring, and increased with epizootics of the disease in host populations in summer and autumn.P. rapae GV disease was prevalent during September and October in populations of P. rapae larvae in plots in which substantial concentrations of the virus were found and contributed to late-season control of the pest insect.     

Immunolocalization of avenin and globulin storage proteins in developing endosperm of Avena sativa L.

The seed storage proteins of oats (Avena sativa L.) are synthesized and assembled into vacuolar protein bodies in developing endosperm tissue. We used double-label immunolocalization to study the distribution of these proteins within protein bodies of the starchy endosperm. When sections of developing oat endosperm sampled 8 d after anthesis were stained with uranyl acetate and lead citrate, the vacuolar protein bodies consisted of light-staining regions which were usually surrounded by a darker-staining matrix. Immunogold staining of this tissue demonstrated a distinct segregation of proteins within protein bodies; globulins were localized in the dark-staining regions and prolamines were localized in the light-staining regions. We observed two additional components of vacuolar protein bodies: a membranous component which was often appressed to the outside of the globulin, and a granular, dark-staining region which resembled tightly clustered ribosomes. Neither antibody immunostained the membranous component, but the granular region was lightly labelled with the anti-globulin antibody. Anti-globulin immunostaining was also observed adjacent to cell walls and appeared to be associated with plasmodesmata. Immunostaining for both antigens was also observed within the rough endoplasmic reticulum. Based on the immunostaining patterns, the prolamine proteins appeared to aggregate within the rough endoplasmic reticulum while most of the globulin appeared to aggregate in the vacuole.Abbreviations DAA days after anthesis - IgG immunoglobulin G - M_r apparent molecular mass - RER rough endoplasmic reticulum - SDS-PAGE sodium dodecyl sulfate — polyacrylamide gel electrophoresis     

Feeding, growth, and the thermal environment of cabbage white caterpillars, Pieris rapae L

Laboratory studies of temperature effects on short-term feeding and growth rates were combined with field data on thermal environments to explore the consequences of temperature variation for growth of caterpillars of the cabbage white butterfly, Pieris rapae. Mean short-term (24-h) consumption and growth rates of fourth-instar P. rapae feeding on collard leaves increased continuously with increasing temperatures between 10 degrees and 35 degrees C, peaked at 35 degrees C, and declined rapidly with temperatures above 35 degrees C. Physical models can mimic temperatures of real fifth-instar caterpillars under collard leaves within 1 degrees -2 degrees C in sunny summer conditions in Seattle, Washington. Continuous recordings of operative temperatures of model caterpillars in a collard garden suggest that, at the timescale of the duration of the fifth instar (5-8 d in the field), P. rapae caterpillars frequently experience temperatures spanning a 25 degrees C range, they spend most of their time at temperatures well below those that maximize growth, and they encounter substantial variation in the frequency distribution of operative temperatures between time periods. Combining these data on growth rate as a function of temperature and the distribution of operative temperatures in the field, I illustrate how growth rates at higher temperatures can make disproportionate contributions to the overall mean growth rates even when higher temperatures are relatively infrequent. Fluctuating thermal conditions may generate variable patterns of selection on reaction norms for growth rate in the field.     

Presence and regulation of trehalose in the body of adult Phormia regina

Trehalose, the major blood sugar of Phormia regina, is present within its tissues in an amount exceeding that in the total blood volume. A major part of the reserve is found in the abdominal fat body. An investigation of trehalose regulation, pursued with the use of a trehalose tolerance test, indicates that within a period of 4 h the adult fly can remove from its blood amounts of this sugar in excess of twice its normal level. The surplus is dealt with in an as yet unknown way, being either sequestered in the tissues (not as trehalose or glucose), metabolized, or excreted in a form other than trehalose or glucose. The process is regulated by the head, and a link between the body and the head must be maintained throughout the entire period of activity.     

Absence of female-specific protein in the hemolymph of stable fly Stomoxys calcitrans (L.) (Diptera: Muscidae)

Changes, during the reproductive cycle, in fat body, hemolymph, and ovarian proteins of the stable fly Stomoxys calcitrans were characterized quantitatively and qualitatively using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein content of all three tissues increased after blood feeding. Fat body protein increased first, followed by hemolymph and ovarian proteins. SDS-PAGE failed to identify vitellogenin in both female hemolymph and fat body samples. No single protein or group of proteins predominated at any stage of the reproductive cycle. Comparisons between male and female stable fly hemolymph and fat body proteins failed to detect female-specific proteins. Female-specific proteins, however, were detected in the hemolymph of four other species of Diptera.     

Structure,development and death of sensory cells and neurons in the pupal labial palp of the butterflies Pieris rapae L. and Pieris brassicae L. (Insecta,Lepidoptera)

Summary The development of neurons possibly related to the outgrowth of axons from the labial palp-pit organ was studied in Pieris rapae. Serial sections of six successive stages between pupation and emergence of the imago were examined with the electron microscope. At pupation the palp contains an apical scolopidial organ (ASO) and cellular strands connected to it. The ASO consists of three type-1 scolopidia, which are characterized by the presence of a ciliary 9 × 2 + 0 pattern throughout the dendritic outer segment and a ciliary dilation beneath the cap. The scolopidia show two special features: (i) the dendritic outer segments reach beyond the cap, and (ii) an intricate junctional complex develops between the dendritic inner segments and the scolopale cells. The cellular strands comprise two types of cells: (1) bipolar cells regarded as neurons due to their cytological features, and (2) enveloping cells, which are wrapped around the bipolar cells. The strands degenerate about 10 h after pupation. The sensory cells of the ASO degenerate consecutively between 28 h and 130 h after pupation. However, their enveloping cells survive and endure in the imago, which emerges about 160 h after pupation. An ASO similarly lacking sensory cells was observed in imagines of Pieris brassicae. It is hypothesized that the ASO and the bipolar neurons of the strands play a role in pathfinding of the axons of the labial palp-pit organ.Supported by the Deutsche Forschungsgemeinschaft (SFB 4/G1)     

Axonal pathfinding in developing labial palps of the butterflies,Pieris rapae and Pieris brassicae

Summary Electron microscopy was used to study the developmental changes in the pupal labial palp of Pieris rapae L. and P. brassicae L. prior to axogenesis of the peripheral receptor organs. Two cells emigrate from the anlage of the labial palp-pit organ (LPPO). They develop growth cones and filopodia, which are directed distally. The filopodia insert into stem cells of the LPPO. The perikarya of the cells proceed into the hemolymph space of the palp such that, eventually, these cells bridge the gap between the LPPO and the apical scolopidial organ (ASO) of the labial palp. This bridge is established at about 11% of total pupal development. Only 6 h later, at 15% of pupal development, the cells are no longer visible. We suggest that these cells are luminal neurons and name them pit-organ luminal (POL) cells. These POL cells may act as primary guiding structures for the axons of the sensory cells of the LPPO, which are assumed to orientate along this structure once they reach the ASO.Supported by the Deutsche Forschungsgemeinschaft (H.A.: SFB4/G1).     

Selectivity in storage hexamerin clearing demonstrated with hemolymph transfusions between Hyalophora cecropia and Actias luna.

When Hyalophora cecropia hemolymph was injected into wandering Actias luna larvae, a methionine-rich hexamerin was selectively transferred to the host's fat body, and completely cleared from the hemolymph by the time of pupal eclosion. Donor arylphorin was 30-40% removed from the hemolymph, and riboflavin-binding hexamerin was even less completely cleared. During the pupal-adult molt, these rates were reversed: methionine-rich hexamerin disappeared no faster than bovine serum albumin, while riboflavin-binding hexamerin was rapidly and completely cleared from the hemolymph, even though A. luna hemolymph lacks a homologue of this protein; arylphorin, again, was cleared at an intermediate rate. Selective clearing of the three hexamerins occurred at similar stages in H. cecropia, their species of origin. Developmentally programmed clearing, with selectivity at least partially conserved between genera, was also demonstrated with transfused vitellogenin: in A. luna females that were forming yolk, H. cecropia vitellogenin was cleared more rapidly than bovine serum albumin; but in younger females, and in males at all stages of metamorphosis, this Mr 510,000 molecule was instead an indicator of nonselective, large protein clearing. Nonselective clearing was more complete during adult development than during pupation. It also showed signs of being more effective for small than for large proteins, insensitive to carbohydrate conjugates, and unsaturated at the protein levels used.     

Major hemolymph proteins from larvae of the black swallowtail butterfly,Papilio polyxenes

Three major hemolymph proteins of Papilio polyxenes larvae were isolated and characterized. Density gradient ultracentrifugation of hemolymph resulted in flotation of the major lipoprotein, lipophorin. P. polyxenes larval lipophorin is composed of two apoproteins, apolipophorin-I and apolipophorin-II, plus a mixture of lipids, to give a density of 1.13 g/ml. Immunoblotting experiments using antisera directed against Manduca sexta apolipophorin-I and apolipophorin-II, respectively, revealed cross-reactivity of apoLp-I with Manduca sexta apoLp-I, and apoLp-II with M. sexta apoLp-II. Gel permeation chromatography of the subnatant obtained following density gradient ultracentrifugation revealed the presence of a major protein peak which was shown to contain three major serum proteins, two of which were isolated and characterized. One of these proteins was purified by lectin affinity chromatography. Both proteins have native molecular weights in the range of 450,000 and appear to be hexamers of a single subunit type. Major serum protein-1 is nonglycosylated and has a subunit molecular weight of 75,000. Major serum protein-2 is glycosylated and has a subunit molecular weight of 74,000. Amino acid analysis of this protein revealed a tyrosine plus phenylalanine content of 20 mole percent, characteristic of the arylphorin class of insect storage proteins. Using antibodies against M. sexta larval hemolymph proteins, both the P. polyxenes major serum proteins were shown to be immunologically related to serum proteins of other lepidopteran species.     

The adaptiveness of searching and host selection behaviour in Pieris rapae (L.)

This study evaluates the adaptive significance of host preferences and searching behaviour in Vancouver and Canberra populations of the cabbage butterfly Pieris rapae (L.). As a result of a complex of responses to plant age, the butterflies concentrate their eggs on middle-aged plants. Young larvae develop faster and survive better on young plants than old ones, but larvae on smaller plants are more susceptible to crowding effects. Thus a preference for plants which are well-grown but not too old is selectively advantageous. By contrast, the butterflies’ host species preferences appear non-adaptive, and are unrelated to the quality of the host as larval food. Vancouver butterflies change their flight direction often and are very responsive to hosts, thereby generating a very aggregated distribution at a low cost in flight time. Canberra butterflies tend to fly in straight lines and are less responsive to hosts; their egg distribution is consequently more nearly random, but they fly further for each egg they lay. The relative costs of aggregation and increased flight time differ between the populations in a manner consistent with the observed behavioural differences.     

Site of synthesis and phylogenetic distribution of a hemolymph trophic factor of the tobacco hornworm,Manduca sexta

Summary Identification of fith instar larvalManduca sexta fat body and epidermis as sites of synthesis of a hemolymph protein (hemolymph trophic factor or HTF) was achieved using in vitro³H-leucine incorporation into protein and subsequent immunoprecipitation of tissue homogenates. Fat body is the primary site of HTF synthesis with a maximal rate on Day 1; epidermis is a secondary site with peak synthesis on Day 0. In vitro radiolabelling followed by TCA precipitation of general protein of fat body and epidermal homogenates suggest that fat body actively elaborates protein on Days 0–5 with peak rates on Days 1 and 4, while epidermis is active on Days 0–5 with a peak rate on Day 3. Based on Anti-HTF ELISA estimates, HTF [500 to 1000 μg/ml] was found in the hemolymph of representatives of the insect orders Blattodea, Hemiptera, Orthoptera, and Lepidoptera and in the class Crustacea, but not in the class Merostomata. These studies suggest a possible fundamental role for HTF among modern arthropods in cuticular deposition involving both epidermis and fat body. The physiological role of HTF is undetermined.     

Uric acid levels during last larval instar of Manduca sexta, an abrupt transition from excretion to storage in fat body

Levels of uric acid in the whole body of the tobacco hornworm, Manduca sexta increased steadily for the 9 days of the fifth instar. However, concentrations in the haemolymph were lowest during the transition from the feeding stage to the wandering stage (days 3, 4), the time when there was a switch from uric acid excretion by the Malpighian tubule-hindgut system to storage in the fat body. Haemolymph volumes, determined for larvae between 2 and 6 days into the fifth instar by isotope dilution with [¹⁴C]-inulin, were used to calculate rates of incorporation of uric acid into Malpighian tubules and fat body of larvae injected with [¹⁴C]-uric acid. These labelling studies indicated that the Malpighian tubules ceased to remove uric acid from the haemolymph some time between the last 6 hr of day 3 of the fifth instar and the first 18 hr of day 4. At the same period, fat body removed significant quantities of uric acid from the haemolymph. The times of initial decreases and increases in levels of uric acid in haemolymph and fat body, respectively, indicated that storage in the fat body started before cessation of elimination via the Malpighian tubule-hindgut system.     

Nutritional and hormonal effects on storage proteins in the silkworm,Bombyx mori L.

Starvation of 48 h old fifth instar larvae depressed storage protein titres initially for 48 h but retained the levels comparable to control thereafter, possibly due to nutrients obtained during the 48 h feeding after fourth ecdysis. After an initial decline ligated larvae accumulated maximum storage proteins in haemolymph. This is because of inhibitory juvenile hormone titre at the basal level besides the appropriate release of 20-hydroxyecdysone from the ectopic source(s). Injection of methoprene (10 Μg/larva) repressed accumulation of storage proteins while 20-hydroxyecdysone (10 Μg/larva) increased the same. P-soyatose injection to starved and ligated larvae accelerated storage protein accumulation in haemolymph, signalling nutrient indispensability for initiation of storage protein synthesis at the appropriate time of last instar development inBombyx mori.