Ultrastructure of the larval Malpighian tubules in <Emphasis Type="Italic">Terrobittacus implicatus</Emphasis> (Mecoptera: Bittacidae)

The larvae of Bittacidae, a cosmopolitan family in Mecoptera, have an interesting habit of spraying the body surface with soil through the anus after hatching, and each molts. The fine structure of Malpighian tubules, however, remains largely unknown in the larvae of Bittacidae to date. Here, we studied the ultrastructure of the larval Malpighian tubules in the hangingfly Terrobittacus implicatus (Huang & Hua) using scanning and transmission electron microscopy. The larvae of T. implicatus have six elongate Malpighian tubules at the junction of the midgut and hindgut. The tubule comprises a basal lamina, a single-layered epithelium, and a central lumen. The basal plasma membranes of the epithelial cells are conspicuously infolded and generate a labyrinth. The epithelium consists of two types of cells: large principal cells and scattered stellate cells. Mitochondria and cisterns of rough endoplasmic reticulum are numerous in the principal cells but are sparsely distributed in the stellate cells, indicating that the principal cells are active in transport. On the other hand, spherites are only abundant in the principal cells and are likely associated with the soil-spraying habit of the larvae.     

Metabolic profiling of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> metamorphosis: a new insight into the central metabolic pathways

Introduction

Metamorphosis is a complicated process in which cell proliferation, differentiation, and death are orchestrated to form the mature structures of insects. In Drosophila, this process is controlled by ecdysone, a steroid hormone responsible for tissue remodeling and organogenesis that gives rise to the adult fly.

Objective

By using a metabolomics approach, this study aimed to elucidate global changes in the central metabolic pathways in Drosophila throughout metamorphosis and then further examine the effects of temperature and origin on metabolic profiles.

Methods

Targeted and non-targeted metabolic profiling of time-course samples from Drosophila were constructed to cover a wide range of cellular metabolites during metamorphosis.

Results

This was the first wide-scale metabolomics study of Drosophila metamorphosis focusing on central metabolism. The abundance of detected metabolites changed drastically and correlated strongly with the development of Drosophila pupae. In non-stress conditions, temperature affected the developmental time, but the metabolic state at a certain stage of metamorphosis remained stable. Between D. melanogaster Canton S and Oregon R, similar metabolic profiles throughout metamorphosis was observed. However, there were still differences in purine and pyrimidine metabolism at an early stage in the pupal period, which was matched by differences in ecdysteroid levels.

Conclusion

This study supported the strength of metabolomics in the field of developmental biology. The results provided a general view on the metabolic profile of Drosophila during metamorphosis, which provides basic 3 knowledge for future metabolomics studies using Drosophila.

     

The initial analysis of a serine proteinase gene (<Emphasis Type="Italic">AccSp10</Emphasis>) from <Emphasis Type="Italic">Apis cerana cerana</Emphasis>: possible involvement in pupal development,innate immunity and abiotic stress responses

Serine proteinases play important roles in innate immunity and insect development. We isolated a serine proteinase gene, designated AccSp10, from the Chinese honeybees (Apis cerana cerana). RT-qPCR and a Western blot analysis at different pupal development stages indicated that AccSp10 might be involved in melanin formation in pupae and promote pupal development. In adult workers, the expression of AccSp10 was upregulated by treatments mimicking harmful environments such as the presence of Bacillus bombysepticus, different temperatures (4, 24 and 42 °C), HgCl₂, H₂O₂ and paraquat; the exception was treatment with VC (vitamin C), which did not upregulate AccSp10 expression. Western blot confirmed the results. A disc diffusion assay indicated that recombinant AccSp10 accelerated E. coli cell death during stimulation with harmful substances (HgCl₂, paraquat and cumene hydroperoxide). These findings suggest that AccSp10 may be involved in the pupal development of Chinese honeybees and protection against microorganisms and abiotic harms.     

小地老虎变态期间马氏管超微结构与酯酶活性的变化

本实验用光镜和电镜观察了小地老虎Agrotis ypsilon Rottemberg幼虫在变态期间马氏管超微结构的变化及成虫马氏管的重组过程,同时还研究了变态期马氏管酯酶的活性.结果表明:(1)变态期间马氏管外形完整,除至预蛹期隐肾复合体解体外,其余无明显变化.(2)变态期间管壁细胞变化显著.幼虫6龄末期马氏管细胞结构开始变化,主要特点为:细胞质电子密度高,充满了核糖体颗粒,微绒毛萎缩,线粒体从萎缩的微绒毛中退出进入细胞质,基膜内褶破坏.进入预蛹期幼虫马氏管细胞解体:基膜内褶、顶端微绒毛、线粒体及细胞质内的其它细胞器消失,并形成自体吞噬泡,细胞质内仅存细胞核及各种类型的液泡.但是在变态期间因底膜始终存在,故马氏管外形不变;至蛹后期,成虫马氏管细胞在原位重组,基膜内褶由浅变深,微绒毛由短变长,线粒体内嵴从无到有.(3)变态过程中羧酸酯酶和酸性磷酸酯酶的活性变化趋势基本相同,以六龄幼虫最强,预蛹期次之,蛹期最低.     

Molecular genetic analysis of five extant reserves of black honeybee <Emphasis Type="Italic">Apis melifera melifera</Emphasis> in the Urals and the Volga region

Local populations of the black honeybee Apis mellifera mellifera from the Urals and the Volga region were examined in comparison with local populations of southern honeybee subspecies A. m. caucasica and A. m. carpatica from the Caucasus and the Carpathians. Genetic analysis was performed on the basis of the polymorphism of nine microsatellite loci of nuclear DNA and the mtDNA COI–COII locus. On the territory of the Urals and the Volga region, five extant populations (reserves) of the black honeybee A. m. mellifera were identified, including the Burzyanskaya, Tatyshlinskaya, Yuzhno-Prikamskaya, Visherskaya, and Kambarskaya populations. These five populations are the basis of the modern gene pool of the black honeybee A. m. mellifera from the Urals and the Volga region. The greatest proportion of the remaining indigenous gene pool of A. m. mellifera (the core of the gene pool of the population of A. m. mellifera) is distributed over the entire territory of Perm krai and the north of the Republic of Bashkortostan. For the population of A. m. mellifera from the Urals and the Volga region, the genetic standards were calculated, which will be useful for future population studies of honeybees.     

Parasitoids and fungal pathogens of <Emphasis Type="Italic">Phyllonorycter issikii</Emphasis> (Kumata, 1963) from Bulgaria

The lime leaf miner, Phyllonorycter issikii, is a moth species native to East Asia, which recently invaded considerable part of Europe. A study on the natural enemies of this invasive species was conducted in Bulgaria. Tilia cordata leaves infested with the lime leaf miner were collected in 2015 and 2016 in two public parks in Sofia. A total of eleven eulophid parasitoids (Hymenoptera: Eulophidae) belonging to three subfamilies - Entedoninae, Eulophinae and Tetrastichinae, and one braconid species (Hymenoptera: Braconidae) were recorded. In addition, one unidentified ichneumonid species was reared. Minotetrastichus platanellus was the most abundant species in the samples comprising 76.88% of the parasitoids reared from collected immature and pupal stages of Ph. issikii. It is followed by Sympiesis gordius (6.53%) and S. sericeicornis (6.03%). Three new host-parasitoid associations were established. The entomopathogenic fungi Beuveria bassiana and Metarhizium anisopliae (Hypocreales) were isolated for the first time from cadavers of Ph. issikii larvae and pupae. Based on the presented observation we hypothesized that the lime leaf miner can produce three generations in Bulgaria.     

Artificial transfer of a thelytoky-inducing <Emphasis Type="Italic">Wolbachia</Emphasis> endosymbiont between strains of the endoparasitoid wasp <Emphasis Type="Italic">Asobara japonica</Emphasis> (Hymenoptera: Braconidae)

Infection with Wolbachia is known to induce diploidization of haploid eggs and enables the production of females from unfertilized eggs. Although there have been several attempts to achieve the artificial horizontal transfer of thelytoky-inducing Wolbachia in parasitoid wasps, the artificial induction of thelytoky has generally been unsuccessful. In this study, we used two strains of Asobara japonica as study materials—one infected with thelytoky-inducing Wolbachia and the other not. We investigated methods of artificially inducing thelytoky by transferring thelytoky-inducing Wolbachia from wasps of the infected strain (the donor wasps) to wasps that had been cured of Wolbachia and to wasps of the uninfected strain (the recipient wasps). To examine the efficiencies of various methods of transfection, we compared the survival and infection rates of recipient wasps that received microinjections at the pupal and adult stages and in different body parts. We also examined the infection rate of the recipients due to cannibalism of Wolbachia-infected pupae. Among those methods, only microinjection at the adult stage resulted in the successful artificial horizontal transfer of Wolbachia, and some of the Wolbachia-infected wasps showed incomplete thelytoky. A low Wolbachia titer in the artificially infected wasps may explain why the thelytoky was incomplete.     

Characteristics and expression of genes encoding two small heat shock protein genes lacking introns from <Emphasis Type="Italic">Chilo suppressalis</Emphasis>

Small heat shock proteins (sHSPs) constitute a large, diverse, and functionally uncharacterized family of heat shock proteins. To gain insight regarding the function of sHSPs in insects, we identified genes encoding two sHSPs, Cshsp22.9b and Cshsp24.3, from the rice pest Chilo suppressalis. The cDNAs of Cshsp22.9b and Cshsp24.3 encoded proteins of 206 and 216 amino acids with isoelectric points of 5.79 and 9.28, respectively. Further characterization indicated that both Cshsp22.9b and Cshsp24.3 lacked introns. Real-time quantitative PCR indicated that Cshsp22.9b and Cshsp24.3 were expressed at higher levels within the fat body as compared to other tissues (head, epidermis, foregut, midgut, hindgut, Malpighian tubules, and hemocytes). Expression of Cshsp22.9b and Cshsp24.3 was lowest in the hindgut and Malpighian tubules, respectively. Cshsp22.9b and Cshsp24.3 showed identical patterns in response to thermal stress from ?11 to 43 °C, and both genes were up-regulated by hot and cold temperatures. The mRNA (messenger ribonucleic acid) expression levels of Cshsp22.9b (KY701308) and Cshsp24.3 (KY701309) were highest after a 2-h exposure at 39 °C and started to decline at 42 °C. In response to cold temperatures, both Cshsp22.9b and Cshsp24.3 showed maximal expression after a 2-h exposure to ?3 °C. The two Cshsps were more responsive to hot than cold temperature stress and were not induced by mildly cold or warm temperatures. In conclusion, Cshsp22.9b and Cshsp24.3 could play a very important role in the regulation of physiological activities in C. suppressalis that are impacted by environmental stimuli.     

Molecular analysis and bioactivity of luteinizing hormone from Japanese eel, <Emphasis Type="Italic">Anguilla japonica</Emphasis>, produced in silkworm pupae

Luteinizing hormone (LH), a gonadotropin hormone (GTH) of the pituitary glycoprotein family, is important in oocyte maturation, ovulation, and spermiation. In this study, we generated a Japanese eel LH (JeLH) with and without the equine chorionic gonadotropin (eCG) carboxyl-terminal peptides under the control of the polyhedron in the silkworm pupae BES to determine their expression levels, glycosylation profile, and in vitro bioactivity. The target proteins were highly expressed in the pupae hemolymph. Recombinant JeLH·eCG and JeLH were N- or O-glycosylated, as shown by periodic-acid Schiff staining, deglycosidase enzyme treatment, and lectin blot analyses, and showed no significant difference in the in vitro bioactivity. Both single-chain hormones and salmon pituitary extract induced maturation of Japanese eel oocytes in vitro. Recombinant LHs produced in silkworm pupae might be suitable candidates for in vivo experiments, because they can be produced in sufficient amount and can undergo N-glycosylation.     

Features of the structure of hymenoptera associated with miniaturization: 1. Anatomy of the fairyfly <Emphasis Type="Italic">Anaphes flavipes</Emphasis> (Hymenoptera,Mymaridae)

Anatomy of the adult fairyfly Anaphes flavipes (Hymenoptera, Mymaridae) is described in detail for the first time, based on series of sections and 3D computer reconstruction. Despite its tiny size, A. flavipes does not demonstrate strong morphological simplification, except for the merging of certain skeletal elements, a slight simplification of endoskeleton, lack of midgut muscles, a reduced number of Malpighian tubules, simplification of the tracheal and circulatory systems, asymmetry of the central nervous system, and the absence of several muscles.     

Effect of <Emphasis Type="Italic">BBX-B8</Emphasis> overexpression on development,body weight,silk protein synthesis and egg diapause of <Emphasis Type="Italic">Bombyx mori</Emphasis>

Bombyxin (BBX) is an insulin-like peptide exists in the silkworm Bombyx mori. Our previous studies on the effects of inhibiting BBX-B8 expression found that BBX-B8 is important for the development of organ, reproduction and trehalose metabolism in the silkworms. In this paper, we investigated the expression profile of the BBX-B8 gene and effect of BBX-B8 overexpression on the development, body weight, silk protein synthesis and egg diapause of B. mori to further understand BBX-B8 functions. BBX-B8 gene expression could be detected in the brains, midguts, anterior silkglands, ovaries, testes, fat bodies, hemolymph, malpighian tubules and embryos by RT-PCR, however it was mainly expressed in the brain. Western blots showed that the change in BBX-B8 expression was not obvious in the brain of 1- to 4-day-old larvae of fifth instar silkworms, but expression increased substantially at 5- to 6-day-old larvae of fifth instar silkworms. Transgenic silkworms overexpressing BBX-B8 were obtained by introducing non-transposon transgenic vector pIZT-B8 containing a BBX-B8 gene driven by Orgyia pseudotsugata nucleopolyhedrovirus IE2 promoter into the genome. Development duration of the transgenic silkworms was delayed by 2.5–3.5 days. Cocoon shell weight of transgenic silkworms was reduced by 4.79 % in females and 7.44 % in males, pupal weight of transgenic silkworms was reduced 6.75 % in females and 13.83 % in males compared to non-transgenic silkworms, and 5.56–14.29 % of transgenic moths laid nondiapausing eggs. All results indicated that BBX-B8 plays an important role in the development, silk protein synthesis and egg diapause of silkworm.     

<Emphasis Type="Italic">Bactrocera oleae</Emphasis> pupae predation by <Emphasis Type="Italic">Ocypus olens</Emphasis> detected by molecular gut content analysis

The pupae of Bactrocera oleae (Diptera: Tephritidae) complete their development during autumn and winter in the soil, rather than in the drupe, resulting susceptible to edaphic predators. Environmentally friendly methods to control this olive pest involve the identification of its natural enemies. This study evaluated the role of Ocypus olens (Coleoptera: Staphylinidae) in the predation of B. oleae pupae, by means of molecular gut content analysis. Modified dry pitfall traps were used to collect live specimens from low-input olive orchards in Tuscany (Italy). Sampling was fine-tuned with a degree-day model estimating the presence of pest pupae in the soil. PCR analyses carried out on field-collected specimens demonstrated that O. olens is a predator of B. oleae, at least during autumn. These results are consistent with predictions of the degree-day model. Knowledge on species composition, traits and complementarity of the natural enemies of B. oleae pupae needs further investigation to advance conservation biological control strategies.     

Effect of excretory-secretory products of some fouling organisms on settling and metamorphosis of the larvae of <Emphasis Type="Italic">Styela rustica</Emphasis> (Ascidiae)

The effect of the excretory-secretory products of some fouling animals on the settling and metamorphosis of larvae of the solitary ascidian Styela rustica was assessed. The substances secreted by the sponge Halichondria panicea stimulated settling of larvae, but concurrently blocked their metamorphosis. The excretory-secretory products of the mussel Mytilus edulis and the ascidian Molgula citrine did not affect settling of the S. rustica larvae but impeded their subsequent development. Water conditioned by the bivalve Hiatella arctica, stimulated settling and, apparently, metamorphosis of the larvae of S. rustica. The chemical substances produced by adult individuals of S. rustica facilitated settling of conspecific larvae but slightly delayed their metamorphosis.     

Sequence of the <Emphasis Type="Italic">mrjp3</Emphasis> Microsatellite Locus in Honeybees of Different Origin

The sequencing of the nucleotide sequences of the mrjp3 repetitive region (mrjp3 microsatellite locus) in Siberian honeybees was carried out. A high similarity of the studied nucleotide sequences (≥99% identity) with reference sequences was observed, which indicates a high conservation of the mrjp3 repetitive region in different Apis mellifera subspecies.     

Features of the structure of hymenoptera associated with miniaturization: 2. Anatomy of <Emphasis Type="Italic">Trichogramma evanescens</Emphasis> (Hymenoptera,Trichogrammatidae)

Anatomy of adults of Trichogramma evanescens (Hymenoptera: Trichogrammatidae) is described in detail for the first time based on series of sections and 3D computer reconstruction. The complex structure of the exoskeleton and musculature (except for reductions of individual muscles) is preserved in T. evanescens despite its tiny size. Considerable simplification is observed in the structure of the intestine, tracheal and circulatory systems, and in the reduced number of Malpighian tubules. Thus, the smallest Hymenoptera (Mymaridae and Trichogrammatidae) demonstrate both the effects of miniaturization universal for insects (reduction of the head endoskeleton, circulatory and respiratory systems) and specific ones.     

On distribution and ecology of some species of cleonines (Coleoptera,Curculionidae): IV. Genus <Emphasis Type="Italic">Lixus</Emphasis> F., subgenus <Emphasis Type="Italic">Eulixus</Emphasis> Reitt.

Data on habitats, food plants of larvae and adults, feeding, mating, oviposition, larval and pupal development, natural enemies, and distribution of seven weevil species (Lixus canescens F.-W., L. iridis Ol., L. myagri Ol., L. punctirostris Boh., L. subtilis Boh., L. incanescens Boh., and L. brevipes Bris.) are given. New host plants of L. canescens and L. iridis are revealed. Gall induction by L. brevipes is reported for the first time. Distribution of all the species in Ukraine and Russia (the latter based on the literature) are given in more detail. Information on the known and potential economic importance of every species is provided.     

The inner ear of <Emphasis Type="Italic">Protungulatum</Emphasis> (Pan-Euungulata,Mammalia)

We present new anatomical details about the bony labyrinth of Protungulatum based on micro CT-scan investigation of an isolated petrosal bone retrieved at the Puercan locality of Bug Creek Anthills and referred to Protungulatum sp. The exceptional state of preservation of the specimen allowed us to reconstruct the very fine details of the inside of the petrosal bone, including the bony labyrinth, the innervation of the vestibule and the innervation and vasculature of the cochlea. Estimation of the auditory capability of Protungulatum based on cochlear morphology indicate that Protungulatum was specialized for high-frequency hearing, with estimated low frequency limits above 1 KHz. Comparisons with Late Cretaceous non-placental eutherians and with early Tertiary pan-euungulates indicate that the bony labyrinth of Protungulatum is closer in general morphology to Mesozoic forms (low coiling and low aspect ratio of the cochlea, posterior orientation of the common crus, dorsal outpocketing of the cochlear fossula), and shares only a few characters with pan-euungulate and euungulate taxa. Interestingly, the bony labyrinth of Protungulatum also shares some morphological features with South American notoungulates and litopterns recently described from Itaboraí, Brazil. These new observations provide new morphological features of potential phylogenetic interest.     

Development,genetic and cytogenetic analyses of genetic sexing strains of the Mexican fruit fly, <Emphasis Type="Italic">Anastrepha ludens</Emphasis> Loew (Diptera: Tephritidae)

Background

Anastrepha ludens is among the pests that have a major impact on México's economy because it attacks fruits as citrus and mangoes. The Mexican Federal government uses integrated pest management to control A. ludens through the Programa Nacional Moscas de la Fruta [National Fruit Fly Program, SAGARPA-SENASICA]. One of the main components of this program is the sterile insect technique (SIT), which is used to control field populations of the pest by releasing sterile flies.

Results

To increase the efficiency of this technique, we have developed a genetic sexing strain (GSS) in which the sexing mechanism is based on a pupal colour dimorphism (brown-black) and is the result of a reciprocal translocation between the Y chromosome and the autosome bearing the black pupae (bp) locus. Ten strains producing wild-type (brown pupae) males and mutant (black pupae) females were isolated. Subsequent evaluations for several generations were performed in most of these strains. The translocation strain named Tapachula-7 showed minimal effect on survival and the best genetic stability of all ten strains. Genetic and cytogenetic analyses were performed using mitotic and polytene chromosomes and we succeeded to characterize the chromosomal structure of this reciprocal translocation and map the autosome breakpoint, despite the fact that the Y chromosome is not visible in polytene nuclei following standard staining.

Conclusions

We show that mitotic and polytene chromosomes can be used in cytogenetic analyses towards the development of genetic control methods in this pest species. The present work is the first report of the construction of GSS of Anastrepha ludens, with potential use in a future Moscafrut operational program.

     

Intestinal enterobacteria of the hibernating <Emphasis Type="Italic">Apis mellifera mellifera</Emphasis> L. bees

Dynamics of enterobacteria of normal intestinal microflora was studied in Apis mellifera mellifera L. bees hibernating under snow in the Western Urals. The cell numbers (N) of the predominant species Klebsiella oxytoca increased from 10-10⁶ CFU/bee in November 2004 to 10⁴-10⁷ CFU/bee in March 2005; its frequency of occurrence (P) increased from 92 to 100%. Increase of Providencia rettgeri (11.2004: N up to 10⁶, P 25%; 03.2005: N 10²-10⁶, P 80%) was accompanied by the substitution of Morganella morganii (11.2004: N up to 10⁶, P 25%) with Proteus vulgaris (03.2005: N up to 10⁵, P 8%). By spring, Hafnia alvei and Citrobacter sp., which are pathogenic to bees, disappeared (11.2004: N up to 10⁵, P 13 and 10%, respectively). Endophytic species Pantoea agglomerans, Leclecria sp., and other representatives of the “Enterobacter agglomerans” group were present in November and after the first emergence in spring (N up to 10⁵; November: P 15%; April: P 23%). In April, the number of enterobacteria decreased to 10⁵, and P. rettgeri became the predominant species (P 54%) instead of K. oxytoca (P 43%).