Ovarian development and vitellogenesis in the sawfly,Athalia rosae ruficornis Jakovlev (Hymenoptera,Tenthredinidae)

Summary

Ovarian development in Athalia rosae ruficornis Jakovlev (Hymenoptera: Tenthredinidae) is described. Number of nurse cells per egg chamber is most often around 60 (close to 63 according to the 2ⁿ–1 rule), but in many cases it deviates from this number significantly. Two major yolk proteins [vitellins: large (apparent molecular weight 160–170 kD) and small (48–50 kD] were identified by SDS-PAGE. Western blotting and immunochemical detection using polyclonal antibodies prepared against each of the vitellins revealed that adult female but not male (both haploid and diploid) hemolymph contains vitellogenins corresponding to these vitellins. Vitellogenins become detectable in the hemolymph of late pupae, and vitellins one day later in the oocytes of adults. Transplantation of immature ovaries into the adult male abdomen caused not only significant accumulation of vitellins in the oocyte but also appearance of small amounts of hemolymph vitellogenins in host males. Injection of homogenate of immature ovaries also caused appearance of small amounts of hemolymph vitellogenins in host males.     

Development of male-incubated ovaries in the gypsy moth,Lymantria dispar

Ovaries from Lymantria dispar females were transplanted into an environment lacking vitellogenin, the male milieu, in order to determine how the presence of vitellogenin in the hemolymph affects the process of protein uptake by gypsy moth oocytes. When undeveloped ovaries from newly ecdysed last instar females were transplanted into males of the same stage, follicles detached from the germarium and increased in size, but the growth of oocytes proceeded more slowly than those from female controls. Although chorion fromation was delayed in male-grown ovaries, scanning electron microscopy of chorionated eggs recovered from adult males showed that a chorion with normal surface architecture was formed by the adult stage. SDS-PAGE analysis of the male-grown ovaries and hemolymph from males receiving ovaries showed that vitellogenin production was not stimulated by the organ transplant and only male hemolymph proteins were internalized by the male-incubated ovaries. Thus, in the absence of vitellogenin, endocytosis of male hemolymph proteins occurred, but the rate of oocyte growth was slowed.     

Occurrence of vitellogenin in drone honeybees (Apis mellifica)

Summary

The occurrence of vitellogenin in adult haploid drones of the honeybee, Apis mellifica, was determined by sensitive immunotechniques, i.e. two-dimensional Immunoelectrophoresis and SDS-PAGE immunoblotting using a monospecific anti-vitellogenin-serum. In drones vitellogenin is one of the minor fractions of the hemolymph proteins. Genetic and regulatory aspects of vitellogenin synthesis in male bees are discussed.     

Identification and characterization of a novel postlarval hemolymph protein from Manduca sexta

The identification, purification and characterization of a new postlarval specific hemolymph protein from Manduca sexta is described. Incorporation of [³⁵S]methionine into Manduca sexta hemolymph proteins in vivo was investigated as a function of development. A major protein band of M_r ≈ 50,000 was highly labeled during the prepupal and adult stage but not in feeding larvae. This postlarval protein (PLP) was isolated from adult male hemolymph and its chemical and immunological properties determined. PLP is a basic protein (pI ～8.6). Electrophoresis under denaturing conditions reveals a subunit M_r ≈ 50,000 while the native protein has an apparent M_r ～ 85,000 by gel permeation chromatography. Anti-PLP serum recognized PLP but not other hemolymph proteins on immunoblots. In vitro translation of fat body mRNA followed by immunoprecipitation revealed that fat body is the site of PLP synthesis. Quantitation of PLP levels in hemolymph throughout development was performed and suggests PLP may play a role in adult development of M. sexta.     

A proteomics approach for the analysis of hemolymph proteins involved in the immediate defense response of the soft tick, <Emphasis Type="Italic">Ornithodoros savignyi</Emphasis>, when challenged with <Emphasis Type="Italic">Candida albicans</Emphasis>

A proteomics approach was employed to identify proteins secreted into the hemolymph of Ornithodorus savignyi ticks 2 h after immune-challenge with the yeast, Candida albicans. Profiling of the proteins present in hemolymph of unchallenged ticks versus ticks challenged with heat-killed yeast revealed five proteins to be differentially expressed. The modulated protein spots were subjected to tandem mass spectrometry (MS/MS) analysis, but could not be positively identified. These proteins can be assigned to the immune response as they were not induced after aseptic injury. In an attempt to identify hemolymph proteins that recognize and bind to yeast cells, hemolymph obtained from both unchallenged and challenged ticks was incubated with C. albicans. Elution of the bound proteins followed by SDS–PAGE analysis indicated that three proteins (97, 88 and 26 kDa) present in both unchallenged and challenged hemolymph samples bind to yeast cells. The constant presence of these three proteins in tick hemolymph leads us to believe that they may be involved in non-self recognition and participate in yeast clearance from tick plasma. The analyzed yeast-binding proteins could also not be positively identified, suggesting that all the tick immune proteins investigated in this study are novel.     

Comparative assessment of the thermal tolerance of spotted stemborer,Chilo partellus (Lepidoptera: Crambidae) and its larval parasitoid,Cotesia sesamiae (Hymenoptera: Braconidae)

Under stressful thermal environments, insects adjust their behavior and physiology to maintain key life‐history activities and improve survival. For interacting species, mutual or antagonistic, thermal stress may affect the participants in differing ways, which may then affect the outcome of the ecological relationship. In agroecosystems, this may be the fate of relationships between insect pests and their antagonistic parasitoids under acute and chronic thermal variability. Against this background, we investigated the thermal tolerance of different developmental stages of Chilo partellus Swinhoe (Lepidoptera: Crambidae) and its larval parasitoid, Cotesia sesamiae Cameron (Hymenoptera: Braconidae) using both dynamic and static protocols. When exposed for 2 h to a static temperature, lower lethal temperatures ranged from ?9 to 6 °C, ?14 to ?2 °C, and ?1 to 4 °C while upper lethal temperatures ranged from 37 to 48 °C, 41 to 49 °C, and 36 to 39 °C for C. partellus eggs, larvae, and C. sesamiae adults, respectively. Faster heating rates improved critical thermal maxima (CT_max) in C. partellus larvae and adult C. partellus and C. sesamiae. Lower cooling rates improved critical thermal minima (CT_min) in C. partellus and C. sesamiae adults while compromising CT_min in C. partellus larvae. The mean supercooling points (SCPs) for C. partellus larvae, pupae, and adults were ?11.82 ± 1.78, ?10.43 ± 1.73 and ?15.75 ± 2.47, respectively. Heat knock‐down time (HKDT) and chill‐coma recovery time (CCRT) varied significantly between C. partellus larvae and adults. Larvae had higher HKDT than adults, while the latter recovered significantly faster following chill‐coma. Current results suggest developmental stage differences in C. partellus thermal tolerance (with respect to lethal temperatures and critical thermal limits) and a compromised temperature tolerance of parasitoid C. sesamiae relative to its host, suggesting potential asynchrony between host–parasitoid population phenology and consequently biocontrol efficacy under global change. These results have broad implications to biological pest management insect–natural enemy interactions under rapidly changing thermal environments.     

Changes in the hemolymph lipophorin and very high density lipoprotein levels during the fifth nymphal and adult stages of Triatoma infestans

The occurrence of lipophorin (HDLp) and a very high density lipoprotein (VHDL) throughout the last nymphal and adult stages of male T. infestans was followed by quantitative immunodiffusion using specific antisera derived from the purified lipoproteins of adult male insects. Significant changes on the concentration of these hemolymph lipoproteins (Lps) occurred during the mentioned developmental stages. These changes include the following. A great accumulation of hemolymph proteins and Lps, particularly VHDL, during the nymphal stage. This increase reached a peak just before the last molt, where the concentration of VHDL and HDLp were 74 and 42 mg/ml hemolymph, respectively. A sharp decline in the Lps concentration just after the molt and during the first 2 weeks of adult life. These changes imply a six-fold decrease on the VHDL and a two-fold decrease in the HDLp concentrations. Another increase in the protein concentration that begins around the third week of the adult stage affecting both Lps, but mainly the HDLp. From its hexameric structure, amino acid composition and high concentration in nymphal stages, the VHDL of T. infestans could be considered a storage protein. The fact that VHDL persists in a considerable concentration in the hemolymph of adult insects differenciates this VHDL from this protein. The distribution of ¹⁴C free fatty acid (FFA) among the hemolymph proteins of T. infestans shows that the FFA are associated only with VHDL and HDLp. The developmental changes of the Lps pattern are accompanied by changes in the relative distribution of FFA between these Lps. The VHDL is the principal carrier of FFA during the fifth-nymph stage while HDLp is the main protein and FFA-carrier in adult life.     

Consequences of diapause on post‐diapause development,reproductive physiology and population growth of Chilo partellus (Swinhoe)

We investigate the effects of diapause on post‐diapause development, reproductive physiology and population growth of Chilo partellus (Swinhoe) (Crambidae: Lepidoptera). Aestivating and hibernating larvae of C. partellus are exposed to diapause terminating conditions (consisting of an LD 12 : 12 h photocycle at 27 ± 1 °C and 65 ± 5% relative humidity with a fresh diet) to terminate the diapause and observations are made on percentage pupation, pupal duration and weight, adult reproductive performance and population growth parameters. We find that the diapause in C. partellus significantly reduces the percentage pupation and weights of pupae, ultimately lowering the weight and reproductive performance of adults. Reduced weights of adult females are found to be directly associated with a lower number of egg cells in ovaries. Nevertheless, the reproductive performance of C. partellus males is also found to be greatly affected in the diapause (hibernation and aestivation) experiencing population in terms of the deposition of a lower number of spermatophores and eupyrene sperm in the reproductive tracts of females compared with the nondiapausing population. The results of the present study clearly indicate that a reduction in longevity, fecundity and egg viability, as well as a reduced rate of deposition of spermatophores and eupyrene sperm, in a diapause experiencing population of C. partellus ultimately leads to a reduction in population growth parameters, thus having implications for bio‐ecology and population dynamics under a changing climatic scenario.     

The <Emphasis Type="Italic">Arabidopsis</Emphasis> LHP1 protein is a component of euchromatin

The HP1 family proteins are involved in several aspects of chromatin function and regulation in Drosophila, mammals and the fission yeast. Here we investigate the localization of LHP1, the unique Arabidopsis thaliana HP1 homolog known at present time, to approach its function. A functional LHP1–GFP fusion protein, able to restore the wild-type phenotype in the lhp1 mutant, was used to analyze the subnuclear distribution of LHP1 in both A. thaliana and Nicotiana tabacum. In A. thaliana interphase nuclei, LHP1 was predominantly located outside the heterochromatic chromocenters. No major aberrations were observed in heterochromatin content or chromocenter organization in lhp1 plants. These data indicate that LHP1 is mainly involved in euchromatin organization in A. thaliana. In tobacco BY-2 cells, the LHP1 distribution, although in foci, slightly differed suggesting that LHP1 localization is determined by the underlying genome organization of plant species. Truncated LHP1 proteins expressed in vivo allowed us to determine the function of the different segments in the localization. The in foci distribution is dependent on the presence of the two chromo domains, whereas the hinge region has some nucleolus-targeting properties. Furthermore, like the animal HP1β and HP1γ subtypes, LHP1 dissociates from chromosomes during mitosis. In transgenic plants expressing the LHP1–GFP fusion protein, two major localization patterns were observed according to cell types suggesting that localization evolves with age or differentiation states. Our results show conversed characteristics of the A. thaliana HP1 homolog with the mammal HP1γ isoform, besides specific plant properties.     

Distribution,relative importance,and agro-climatic preferences of cereal stem borers in Eastern Ethiopia

Abstract

The distribution and relative importance of lepidopteran stem borers attacking maize and sorghum were investigated in farmers’ fields during the main cropping seasons of 2015 and 2016 across three main agro-climatic zones (ACZs) of eastern Ethiopia. Three stem borer species, namely Busseola fusca Fuller, Chilo partellus Swinhoe and Sesamia calamistis Hampson were found attacking these crops with different levels of distribution and abundance among ACZs. Busseola fusca was the only borer species in the highlands and dominant (67%) in the midhighland zones, while C. partellus was predominant (75%) in the lowlands. The abundance of S. calamistis was low across its distribution range. Across both years, mean plant infestation by B. fusca ranged from 20.38 in lowlands to 42.97% in highlands. Chilo partellus resulted in a mean infestation of 27.38% in the midaltitude to 68.24% in lowlands. Mean density of larvae per plant was higher for C. partellus (2.85), followed by B. fusca (1.41) and S. calamistis (0.46). Moreover, within each ACZs, variation in plant damage variables was observed between the study periods. These spatio-temporal differences in community structures of stem borers are discussed particularly in relation to agro-climatic requirements.     

Nuclear binding sites for juvenile hormone III in the male accessory reproductive glands of Melanoplus sanguinipes

Summary

We have identified a potential nuclear juvenile hormone (JH) receptor in the long hyaline tubules (LHT), part of the male accessory reproductive gland (MARG) of M. sanguinipes. The MARG was incubated in vitro with [³H]JH III, and the distribution of the [³H]JH III among the cellular fractions of the LHT was determined. Some 37±4% of the radioactivity was associated with the crude nuclear pellet, while the cytosolic, microsomal and mitochondrial fractions contained 30±3%, 23±2% and 10±1%, respectively. The bound JH III was measured in nuclear extracts of LHT from males up to 15 days post-eclosion. These results revealed that JH binding increased in an age-dependent manner up to day 7, then levelled off to day 12, to increase again on day 14. The nuclear-binding component in the LHT had a very strong affinity for JH III, with a K_D value of 0.8 nM. Our observations are considered in relation to the potential site and mode of action of JH.     

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.     

Synergistic activity between Bacillus thuringiensis Cry1Ab and Cry1Ac toxins against maize stem borer (Chilo partellus Swinhoe)

Aim: To select a toxin combination for the management of maize stem borer (Chilo partellus) and to understand possible mechanism of synergism among Bacillus thuringiensis Cry1A toxins tested. Methods and Results: Three Cry1A toxins were over expressed in Escherichia coli strain JM105 and used for diet overlay insect bioassay against C. partellus neonate larvae, both alone and in combinations. Probit analysis revealed that the three Cry1A toxins tested have synergistic effect against C. partellus larvae. In vitro binding analysis of fluorescein isothiocyanate (FITC)‐labelled Cry1A toxins to midgut brush border membrane vesicle (BBMV) shows that increase in toxicity is directly correlated to an increase in binding of toxin mix. Conclusions: A high Cry1Ac to Cry1Ab ratio leads to an increase in efficacy of these toxins towards C. partellus larvae and this increase in toxicity comes from an increase in toxin binding. Significance and Impact of the Study: Use of Cry1Ab and Cry1Ac combination could be an effective approach to control C. partellus. Furthermore, we show it first time that possible reason behind increase in toxicity of synergistic Cry1A proteins is an increase in toxin binding.     

Expression of larval hemolymph proteins (Lhp) genes and protein synthesis in the fat body of greater wax moth (Galleria mellonella) larvae during diapause

When one-day-old, last instar Galleria mellonella larvae are exposed to 18 degrees C they enter diapause and cease further development for several months. During diapause a group of proteins (72-84 kDa) synthesized in the fat body and secreted into the hemolymph is markedly elevated. Partial sequencing of the N-terminus of two proteins from this group confirmed their identity with larval hemolymph proteins (LHP) belonging to the family of hexameric storage proteins. The expression of two Lhp genes of known sequence (Lhp76 and Lhp82) were monitored in both diapausing and non-diapausing individuals. The expression of both genes and subsequent synthesis of the proteins (LHP76 and LHP82) is maintained until at least 90-100 days of diapause.     

Capsicum annuum proteinase inhibitor ingestion negatively impacts the growth of sorghum pest Chilo partellus and promotes differential protease expression

BackgroundChilo partellus is an important insect pest infesting sorghum and maize. The larvae internalize in the stem, rendering difficulties in pest management. We investigated the effects of Capsicum annuum proteinase inhibitors (CanPIs) on C. partellus larvae by in-vitro and in-vivo experiments.MethodsRecombinant CanPI-7 (with four-Inhibitory Repeat Domains, IRDs), -22 (two-IRDs) and insect proteinase activities were estimated by proteinase assays, dot blot assays and in gel activity assays. Feeding bioassays of lab reared C. partellus with CanPI-7 and -22 were performed. C. partellus proteinase gene expression was done by RT-PCR. In-silico structure prediction of proteinases and CanPI IRDs was carried out, their validation and molecular docking was done for estimating the interaction strength.ResultsLarval proteinases of C. partellus showed higher activity at alkaline pH and expressed few proteinase isoforms. Both CanPIs showed strong inhibition of C. partellus larval proteinases. Feeding bioassays of C. partellus with CanPIs revealed a dose dependent retardation of larval growth, reduction of pupal mass and fecundity, while larval and pupal periods increased significantly. Ingestion of CanPIs resulted in differential up-regulation of C. partellus proteinase isoforms, which were sensitive to CanPI-7 but were insensitive to CanPI-22. In-silico interaction studies indicated the strong interaction of IRD-9 (of CanPI-22) with Chilo proteinases tested.ConclusionsOf the two PIs tested, CanPI-7 prevents induction of inhibitor insensitive proteinases in C. partellus so it can be explored for developing C. partellus tolerance in sorghum.General significanceIngestion of CanPIs, effectively retards C. partellus growth; while differentially regulating the proteinases.     

Larval dispersal of the invasive fall armyworm,Spodoptera frugiperda,the exotic stemborer Chilo partellus,and indigenous maize stemborers in Africa

Larval dispersal either through ballooning or crawling results in a redistribution of the insect population and infestations within and between plants. In addition, invasive species, such as the fall armyworm (FAW), Spodoptera frugiperda (JE Smith) (Lepidoptera: Noctuidae), and the exotic stemborer Chilo partellus (Swinhoe) (Lepidoptera: Crambidae), may displace indigenous stemborers on maize in Africa. To test whether larval dispersal activity may play a role in the displacement of indigenous stemborers, larval dispersal was compared between FAW, C. partellus, and the indigenous species Busseola fusca (Fuller) and Sesamia calamistis (Hampson) (both Lepidoptera: Noctuidae). Twenty potted maize plants were infested with one batch of eggs either from stemborers (B. fusca, S. calamistis, or C. partellus) or from FAW and monitored in the greenhouse for ballooning activities. After egg hatching, both ballooning and non-ballooning larvae were identified according to species and counted. FAW neonate larvae had greater potential for ballooning off than stemborers, irrespective of species. For each species, more females dispersed than males, and their survival rate was higher than that of non-ballooning larvae. In addition, plant-to-plant larval movements were studied using 6.25-m² plots of caged maize in a completely randomized design with five replicates. FAW was found to have wider dispersal and plant damage potential than any of the stemborer species. In conclusion, in contrast to C. partellus, the invasive characteristic of FAW can be explained, in part, by its higher larval dispersal activity compared to stemborers. This difference in larval dispersal might also be considered in sampling plans for monitoring pest density in the field.     

Aldehyde oxidase cross-reacting material in the Aldox n,cin, mal,and lxd mutants of Drosophila melanogaster

Rocket immunoelectrophoresis was used to estimate aldehyde oxidase cross-reacting material (AO-CRM) in larval hemolymph and adult fly extracts in mutants with reduced AO enzymatic activity. Hemolymph of larvae homozygous for Aldox ⁿ, which is a mutation of the presumed structural gene for AO, contains 30% of the wild-type CRM. The demonstration of AO-CRM in Aldox ⁿ larval hemolymph is surprising since this genotype has been reported to lack CRM. By contrast, adult Aldox ⁿ flies lack detectable CRM. The other AO-deficient mutants that were examined are cin, mal, and lxd; each has appreciable levels of CRM in both larval hemolymph and adult extracts. Detection of CRM in these mutants helps to clarify conflicting reports in the literature.This research was supported by a grant from the Natural Sciences and Engineering Research Council of Canada to L.W.B.     

Purification and functional characterization of lectin with phenoloxidase activity from the hemolymph of cockroach,Periplaneta americana

Lectins also identified as hemagglutinins are multivalent proteins and on account of their fine sugar‐binding specificity play an important role in immune system of invertebrates. The present study was carried out on the hemolymph lectin of cockroach, Periplaneta americana with appropriate screening and purification to understand its molecular as well as functional nature. The lectin from the hemolymph was purified using ion‐exchange chromatography. The approximate molecular weight of purified lectin was 340 kDa as determined by FPLC analysis. Rabbit erythrocytes were highly agglutinated with purified lectin from the hemolymph of P. americana. The hemagglutination activity (HA) of lectin was specifically inhibited by fucose. Glycoproteins also inhibited the HA activity of lectin. The amino acid sequences of the purified lectin revealed homology with amino acid sequences of allergen proteins from P. americana. Purified lectin showed the highest phenoloxidase activity against dopamine. The activators such as exogenous proteases and LPS from Escherichia coli and Salmonella minnesota significantly enhanced the PO activity of the purified lectin. Besides, the presence of copper and hemocyanin conserved domain in the purified lectin provided a new facet that insects belonging to the ancient clade such as cockroaches retained some traces of evolutionary resemblance in possessing lectin of ancient origin.