Colony sex ratios vary with breeding structure but not relatedness asymmetry in the facultatively polygynous ant Pheidole pallidula

We investigated sex allocation in a Mediterranean population of the facultatively polygynous (multiple queen per colony) ant Pheidole pallidula. This species shows a strong split sex ratio, with most colonies producing almost exclusively a single-sex brood. Our genetic (microsatellite) analyses reveal that P. pallidula has an unusual breeding system, with colonies being headed by a single or a few unrelated queens. As expected in such a breeding system, our results show no variation in relatedness asymmetry between monogynous (single queen per colony) and polygynous colonies. Nevertheless, sex allocation was tightly associated with the breeding structure, with monogynous colonies producing a male-biased brood and polygynous colonies almost only females. In addition, sex allocation was closely correlated with colony total sexual productivity. Overall, our data show that when colonies become more productive (and presumably larger) they shift from monogyny to polygyny and from male production to female production, a pattern that has never been reported in social insects.

Identification of microRNAs expressed in two mosquito vectors, Aedes albopictus and Culex quinquefasciatus.

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression in a variety of organisms, including insects, vertebrates, and plants. miRNAs play important roles in cell development and differentiation as well as in the cellular response to stress and infection. To date, there are limited reports of miRNA identification in mosquitoes, insects that act as essential vectors for the transmission of many human pathogens, including flaviviruses. West Nile virus (WNV) and dengue virus, members of the Flaviviridae family, are primarily transmitted by Aedes and Culex mosquitoes. Using high-throughput deep sequencing, we examined the miRNA repertoire in Ae. albopictus cells and Cx. quinquefasciatus mosquitoes. RESULTS: We identified a total of 65 miRNAs in the Ae. albopictus C7/10 cell line and 77 miRNAs in Cx. quinquefasciatus mosquitoes, the majority of which are conserved in other insects such as Drosophila melanogaster and Anopheles gambiae. The most highly expressed miRNA in both mosquito species was miR-184, a miRNA conserved from insects to vertebrates. Several previously reported Anopheles miRNAs, including miR-1890 and miR-1891, were also found in Culex and Aedes, and appear to be restricted to mosquitoes. We identified seven novel miRNAs, arising from nine different precursors, in C7/10 cells and Cx. quinquefasciatus mosquitoes, two of which have predicted orthologs in An. gambiae. Several of these novel miRNAs reside within a ~350 nt long cluster present in both Aedes and Culex. miRNA expression was confirmed by primer extension analysis. To determine whether flavivirus infection affects miRNA expression, we infected female Culex mosquitoes with WNV. Two miRNAs, miR-92 and miR-989, showed significant changes in expression levels following WNV infection. CONCLUSIONS: Aedes and Culex mosquitoes are important flavivirus vectors. Recent advances in both mosquito genomics and high-throughput sequencing technologies enabled us to interrogate the miRNA profile in these two species. Here, we provide evidence for over 60 conserved and seven novel mosquito miRNAs, expanding upon our current understanding of insect miRNAs. Undoubtedly, some of the miRNAs identified will have roles not only in mosquito development, but also in mediating viral infection in the mosquito host.

Morphological and genomic characterization of Filobasidiella depauperata: a homothallic sibling species of the pathogenic cryptococcus species complex.

The fungal species Cryptococcus neoformans and Cryptococcus gattii cause respiratory and neurological disease in animals and humans following inhalation of basidiospores or desiccated yeast cells from the environment. Sexual reproduction in C. neoformans and C. gattii is controlled by a bipolar system in which a single mating type locus (MAT) specifies compatibility. These two species are dimorphic, growing as yeast in the asexual stage, and producing hyphae, basidia, and basidiospores during the sexual stage. In contrast, Filobasidiella depauperata, one of the closest related species, grows exclusively as hyphae and it is found in association with decaying insects. Examination of two available strains of F. depauperata showed that the life cycle of this fungal species shares features associated with the unisexual or same-sex mating cycle in C. neoformans. Therefore, F. depauperata may represent a homothallic and possibly an obligately sexual fungal species. RAPD genotyping of 39 randomly isolated progeny from isolate CBS7855 revealed a new genotype pattern in one of the isolated basidiospores progeny, therefore suggesting that the homothallic cycle in F. depauperata could lead to the emergence of new genotypes. Phylogenetic analyses of genes linked to MAT in C. neoformans indicated that two of these genes in F. depauperata, MYO2 and STE20, appear to form a monophyletic clade with the MATa alleles of C. neoformans and C. gattii, and thus these genes may have been recruited to the MAT locus before F. depauperata diverged. Furthermore, the ancestral MATa locus may have undergone accelerated evolution prior to the divergence of the pathogenic Cryptococcus species since several of the genes linked to the MATa locus appear to have a higher number of changes and substitutions than their MATalpha counterparts. Synteny analyses between C. neoformans and F. depauperata showed that genomic regions on other chromosomes displayed conserved gene order. In contrast, the genes linked to the MAT locus of C. neoformans showed a higher number of chromosomal translocations in the genome of F. depauperata. We therefore propose that chromosomal rearrangements appear to be a major force driving speciation and sexual divergence in these closely related pathogenic and saprobic species.

A switch in the control of growth of the wing imaginal disks of Manduca sexta.

BACKGROUND: Insulin and ecdysone are the key extrinsic regulators of growth for the wing imaginal disks of insects. In vitro tissue culture studies have shown that these two growth regulators act synergistically: either factor alone stimulates only limited growth, but together they stimulate disks to grow at a rate identical to that observed in situ. It is generally thought that insulin signaling links growth to nutrition, and that starvation stops growth because it inhibits insulin secretion. At the end of larval life feeding stops but the disks continue to grow, so at that time disk growth has become uncoupled from nutrition. We sought to determine at exactly what point in development this uncoupling occurs. METHODOLOGY: Growth and cell proliferation in the wing imaginal disks and hemolymph carbohydrate concentrations were measured at various stages in the last larval instar under experimental conditions of starvation, ligation, rescue, and hormone treatment. PRINCIPAL FINDINGS: Here we show that in the last larval instar of M. sexta, the uncoupling of nutrition and growth occurs as the larva passes the critical weight. Before this time, starvation causes a decline in hemolymph glucose and trehalose and a cessation of wing imaginal disks growth, which can be rescued by injections of trehalose. After the critical weight the trehalose response to starvation disappears, and the expression of insulin becomes decoupled from nutrition. After the critical weight the wing disks loose their sensitivity to repression by juvenile hormone, and factors from the abdomen, but not the brain, are required to drive continued growth. CONCLUSIONS: During the last larval instar imaginal disk growth becomes decoupled from somatic growth at the time that the endocrine events of metamorphosis are initiated. These regulatory changes ensure that disk growth continues uninterrupted when the nutritive and endocrine signals undergo the drastic changes associated with metamorphosis.

Electron tomography of cryofixed, isometrically contracting insect flight muscle reveals novel actin-myosin interactions.

BACKGROUND: Isometric muscle contraction, where force is generated without muscle shortening, is a molecular traffic jam in which the number of actin-attached motors is maximized and all states of motor action are trapped with consequently high heterogeneity. This heterogeneity is a major limitation to deciphering myosin conformational changes in situ. METHODOLOGY: We used multivariate data analysis to group repeat segments in electron tomograms of isometrically contracting insect flight muscle, mechanically monitored, rapidly frozen, freeze substituted, and thin sectioned. Improved resolution reveals the helical arrangement of F-actin subunits in the thin filament enabling an atomic model to be built into the thin filament density independent of the myosin. Actin-myosin attachments can now be assigned as weak or strong by their motor domain orientation relative to actin. Myosin attachments were quantified everywhere along the thin filament including troponin. Strong binding myosin attachments are found on only four F-actin subunits, the "target zone", situated exactly midway between successive troponin complexes. They show an axial lever arm range of 77°/12.9 nm. The lever arm azimuthal range of strong binding attachments has a highly skewed, 127° range compared with X-ray crystallographic structures. Two types of weak actin attachments are described. One type, found exclusively in the target zone, appears to represent pre-working-stroke intermediates. The other, which contacts tropomyosin rather than actin, is positioned M-ward of the target zone, i.e. the position toward which thin filaments slide during shortening. CONCLUSION: We present a model for the weak to strong transition in the myosin ATPase cycle that incorporates azimuthal movements of the motor domain on actin. Stress/strain in the S2 domain may explain azimuthal lever arm changes in the strong binding attachments. The results support previous conclusions that the weak attachments preceding force generation are very different from strong binding attachments.

The adaptive significance and prevalence of courtship feeding in Hawaiian swordtail crickets

Males of many insect species feed their partner during courtship and mating. Studies of male nutrient donation in various systems have established that nuptial feeding has evolved mostly through sexual selection. Although there is extensive diversity in form, the function of nuptial gifts is typically limited to either facilitating copulation or increasing ejaculate transfer, depending on the time at which the gift is consumed by females. Unlike other insects, the Hawaiian swordtail cricket Laupala (Gryllidae: Trigonidiinae) exhibits serial transfer of nuptial gifts. Males transfer multiple spermless 'micro' spermatophores over several hours before mating at the end of the day (i.e. before the transfer of a single sperm-containing 'macro' spermatophore). By experimental manipulation of male microspermatophore donation, I tested several hypotheses pertaining to the adaptive significance of nuptial gifts in this system. I found that microspermatophore transfer improves insemination, by causing the female reproductive tract to take in more sperm. This result reveals a previously undocumented function for premating nuptial gift donation among insects. Enhanced sperm transfer due to microspermatophore donation may represent male manipulation or an internal mechanism of post-copulatory choice by females. I also performed experimental manipulation of male photoperiod to investigate how time and gender influence nuptial gift production and mating behavior. I found that the timing of mating is limited in males but not females and that the time of pair formation has consequences for the degree of nuptial gift donation, which suggests that both mating timing and microspermatophore number is important for male reproductive success. Finally, I observed the mating behavior of several trigonidiine taxa for a comparative analysis of sexual behavior and found that other genera also utilize spermless microspermatophores, which suggests that microspermatophore donation may be a common nuptial gift strategy among swordtail crickets. The elaborate nuptial feeding behavior of Hawaiian swordtail crickets prior to mating represents a newly discovered strategy to increase male insemination success rather than mating success. Based on this unexpected result, it is worth exploring whether courtship behaviors in other cricket or insect mating systems have also evolved to increase sperm uptake.

Impacts of Mountaintop Removal Coal Mining on the Mud River, West Virginia: Selenium Accumulation, Trophic Transfer, and Toxicity in Fish

Selenium (Se) is a micronutrient necessary for the function of a variety of important enzymes; Se also exhibits a narrow range in concentrations between essentiality and toxicity. Oviparous vertebrates such as birds and fish are especially sensitive to Se toxicity, which causes reproductive impairment and defects in embryo development. Selenium occurs naturally in the Earth's crust, but it can be mobilized by a variety of anthropogenic activities, including agricultural practices, coal burning, and mining.

Mountaintop removal/valley fill (MTR/VF) coal mining is a form of surface mining found throughout central Appalachia in the United States that involves blasting off the tops of mountains to access underlying coal seams. Spoil rock from the mountain is placed into adjacent valleys, forming valley fills, which bury stream headwaters and negatively impact surface water quality. This research focused on the biological impacts of Se leached from MTR/VF coal mining operations located around the Mud River, West Virginia.

In order to assess the status of Se in a lotic (flowing) system such as the Mud River, surface water, insects, and fish samples including creek chub (Semotilus atromaculatus) and green sunfish (Lepomis cyanellus) were collected from a mining impacted site as well as from a reference site not impacted by mining. Analysis of samples from the mined site showed increased conductivity and Se in the surface waters compared to the reference site in addition to increased concentrations of Se in insects and fish. Histological analysis of mined site fish gills showed a lack of normal parasites, suggesting parasite populations may be disrupted due to poor water quality. X-ray absorption near edge spectroscopy techniques were used to determine the speciation of Se in insect and creek chub samples. Insects contained approximately 40-50% inorganic Se (selenate and selenite) and 50-60% organic Se (Se-methionine and Se-cystine) while fish tissues contained lower proportions of inorganic Se than insects, instead having higher proportions of organic Se in the forms of methyl-Se-cysteine, Se-cystine, and Se-methionine.

Otoliths, calcified inner ear structures, were also collected from Mud River creek chubs and green sunfish and analyzed for Se content using laser ablation inductively couple mass spectrometry (LA-ICP-MS). Significant differences were found between the two species of fish, based on the concentrations of otolith Se. Green sunfish otoliths from all sites contained background or low concentrations of otolith Se (< 1 µg/g) that were not significantly different between mined and unmined sites. In contrast creek chub otoliths from the historically mined site contained much higher (≥ 5 µg/g, up to approximately 68 µg/g) concentrations of Se than for the same species in the unmined site or for the green sunfish. Otolith Se concentrations were related to muscle Se concentrations for creek chubs (R2 = 0.54, p = 0.0002 for the last 20% of the otolith Se versus muscle Se) while no relationship was observed for green sunfish.

Additional experiments using biofilms grown in the Mud River showed increased Se in mined site biofilms compared to the reference site. When we fed fathead minnows (Pimephales promelas) on these biofilms in the laboratory they accumulated higher concentrations of Se in liver and ovary tissues compared to fathead minnows fed on reference site biofilms. No differences in Se accumulation were found in muscle from either treatment group. Biofilms were also centrifuged and separated into filamentous green algae and the remaining diatom fraction. The majority of Se was found in the diatom fraction with only about 1/3rd of total biofilm Se concentration present in the filamentous green algae fraction

Finally, zebrafish (Danio rerio) embryos were exposed to aqueous Se in the form of selenate, selenite, and L-selenomethionine in an attempt to determine if oxidative stress plays a role in selenium embryo toxicity. Selenate and selenite exposure did not induce embryo deformities (lordosis and craniofacial malformation). L-selenomethionine, however, induced significantly higher deformity rates at 100 µg/L compared to controls. Antioxidant rescue of L-selenomethionime induced deformities was attempted in embryos using N-acetylcysteine (NAC). Pretreatment with NAC significantly reduced deformities in the zebrafish embryos secondarily treated with L-selenomethionine, suggesting that oxidative stress may play a role in Se toxicity. Selenite exposure also induced a 6.6-fold increase in glutathione-S-transferase pi class 2 gene expression, which is involved in xenobiotic transformation. No changes in gene expression were observed for selenate or L-selenomethionine-exposed embryos.

The findings in this dissertation contribute to the understanding of how Se bioaccumulates in a lotic system and is transferred through a simulated foodweb in addition to further exploring oxidative stress as a potential mechanism for Se-induced embryo toxicity. Future studies should continue to pursue the role of oxidative stress and other mechanisms in Se toxicity and the biotransformation of Se in aquatic ecosystems.

A Quantitative Analysis of Growth and Size Regulation in Manduca sexta: The Physiological Basis of Variation in Size and Age at Metamorphosis.

Body size and development time are important life history traits because they are often highly correlated with fitness. Although the developmental mechanisms that control growth have been well studied, the mechanisms that control how a species-characteristic body size is achieved remain poorly understood. In insects adult body size is determined by the number of larval molts, the size increment at each molt, and the mechanism that determines during which instar larval growth will stop. Adult insects do not grow, so the size at which a larva stops growing determines adult body size. Here we develop a quantitative understanding of the kinetics of growth throughout larval life of Manduca sexta, under different conditions of nutrition and temperature, and for genetic strains with different adult body sizes. We show that the generally accepted view that the size increment at each molt is constant (Dyar's Rule) is systematically violated: there is actually a progressive increase in the size increment from instar to instar that is independent of temperature. In addition, the mass-specific growth rate declines throughout the growth phase in a temperature-dependent manner. We show that growth within an instar follows a truncated Gompertz trajectory. The critical weight, which determines when in an instar a molt will occur, and the threshold size, which determines which instar is the last, are different in genetic strains with different adult body sizes. Under nutrient and temperature stress Manduca has a variable number of larval instars and we show that this is due to the fact that more molts at smaller increments are taken before threshold size is reached. We test whether the new insight into the kinetics of growth and size determination are sufficient to explain body size and development time through a mathematical model that incorporates our quantitative findings.

Big or fast: two strategies in the developmental control of body size.

Adult body size is controlled by the mechanisms that stop growth when a species-characteristic size has been reached. The mechanisms by which size is sensed and by which this information is transduced to the growth regulating system are beginning to be understood in a few species of insects. Two rather different strategies for control have been discovered; one favors large body size and the other favors rapid development.

Insectos y un hongo relacionados con el Caraguata (Eryngium Paniculatum)

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Effect of Bacillus thuringiensis naturally colonising Brassica campestris var. chinensis leaves on neonate larvae of Pieris brassicae

The feeding of neonate larvae of Pieris brassicae (Order Lepidoptera) on leaves of brassica plants that had been colonised by Bacillus thuringiensis resulted in the death of 35% of the population within 72 h. The bacteria Multiplied in the cadavers, resulting in an increase of about 50-fold compared to the living insects. Surviving insects showed no ill effects during the time of the study. There was negligible multiplication of B. thuringiensis in the frass. (C) 2009 Elsevier Inc. All rights reserved.

The ecology of Bacillus thuringiensis on the Phylloplane: colonization from soil, plasmid transfer, and interaction with larvae of Pieris brassicae

Seedlings of clover (Triflorium hybridum) were colonized by Bacillus thuringiensis when spores and seeds were co-inoculated into soil. Both a strain isolated in the vegetative form from the phylloplane of clover, 2810-S-4, and a laboratory strain, HD-1, were able to colonize clover to a density of about 1000 CFU/g leaf when seeds were sown in sterile soil and to a density of about 300 CFU/g leaf in nonsterile soil. A strain lacking the characteristic insecticidal crystal proteins produced a similar level of colonization over a 5-week period as the wild type strain, indicating that crystal production was not a mitigating factor during colonization. A small plasmid, pBC16, was transferred between strains of B. thuringiensis when donor and recipient strains were sprayed in vegetative form onto leaves of clover and pak choi (Brassica campestris var. chinensis). The rate of transfer was about 0.1 transconjugants/recipient and was dependent on the plant species. The levels of B. thuringiensis that naturally colonized leaves of pak choi produced negligible levels of mortality in third instar larvae of Pieris brassicae feeding on the plants. Considerable multiplication occurred in the excreted frass but not in the guts of living insects. Spores in the frass could be a source of recolonization from the soil and be transferred to other plants. These findings illustrate a possible cycle, not dependent on insect pathology, by which B. thuringiensis diversifies and maintains itself in nature.

GB Non-native Species Information Portal: documenting the arrival of non-native species in Britain

Information on non-native species (NNS) is often scattered among a multitude of sources, such as regional and national databases, peer-reviewed and grey literature, unpublished research projects, institutional datasets and with taxonomic experts. Here we report on the development of a database designed for the collation of information in Britain. The project involved working with volunteer experts to populate a database of NNS (hereafter called “the species register”). Each species occupies a row within the database with information on aspects of the species’ biology such as environment (marine, freshwater, terrestrial etc.), functional type (predator, parasite etc.), habitats occupied in the invaded range (using EUNIS classification), invasion pathways, establishment status in Britain and impacts. The information is delivered through the Great Britain Non-Native Species Information Portal hosted by the Non-Native Species Secretariat. By the end of 2011 there were 1958 established NNS in Britain. There has been a dramatic increase over time in the rate of NNS arriving in Britain and those becoming established. The majority of established NNS are higher plants (1,376 species). Insects are the next most numerous group (344 species) followed by non-insect invertebrates (158 species), vertebrates (50 species), algae (24 species) and lower plants (6 species). Inventories of NNS are seen as an essential tool in the management of biological invasions. The use of such lists is diverse and far-reaching. However, the increasing number of new arrivals highlights both the dynamic nature of invasions and the importance of updating NNS inventories.

“Islands” in Holocene forests: implications for forest openness, landscape clearance and “culture-steppe” species

Human activity has undoubtedly had a major impact on Holocene forested ecosystems, with the concurrent expansion of plants and animals associated with cleared landscapes and pasture, also known as 'culture-steppe'. However, this anthropogenic perspective may have underestimated the contribution of autogenic disturbance (e.g. wind-throw, fire), or a mixture of autogenic and anthropogenic processes, within early Holocene forests. Entomologists have long argued that the north European primary forest was probably similar in structure to pasture woodland. This idea has received support from the conservation biologist Frans Vera, who has recently strongly argued that the role of large herbivores in maintaining open forests in the primeval landscapes of Europe has been seriously underestimated. This paper reviews this debate from a fossil invertebrate perspective and looks at several early Holocene insect assemblages. Although wood taxa are indeed important during this period, species typical of open areas and grassland and dung beetles, usually associated with the dung of grazing animals, are persistent presences in many early woodland faunas. We also suggest that fire and other natural disturbance agents appear to have played an important ecological role in some of these forests, maintaining open areas and creating open vegetation islands within these systems. More work, however, is required to ascertain the role of grazing animals, but we conclude that fossil insects have a significant contribution to make to this debate. This evidence has fundamental implications in terms of how the palaeoecological record is interpreted, particularly by environmental archaeologists and palaeoecologists who may be more interested in identifying human-environment interactions rather than the ecological processes which may be preserved within palaeoecological records.

Pinus and Prostomis: a dendrochronological and palaeoentomological study of a study of a mid-Holocene woodland in Eastern England

Tree-ring analysis of sub-fossil Pinus sylvestris L. and Quercus sp. and their associated sub-fossil insect assemblages from tree rot holes have been used to study a prehistoric forest buried in the basal peats at Tyrham Hall Quarry, Hatfield Moors SSSI, in the Humberhead Levels, eastern England. The site provided a rare opportunity to examine the date, composition, age structure and entomological biodiversity of a mid-Holocene Pinus-dominated forest. The combined approaches of dendrochronology and palaeoentomology have enabled a detailed picture of the forest to be reconstructed, within a precise time frame. The Pinus chronology has been precisely dated to 2921- 2445 BC against the English Quercus master curve and represents the first English Pinus chronology to be dendrochronologically dated. A suite of important xylophilous (wood-loving) beetles that are today very rare and four species that no longer live within the British Isles were also recovered, their disappearance associated with the decline in woodland habitats as well as possible climate change. The sub-fossil insects indicate that the characteristic species of the site's modern-day fauna were already in place 4000 years ago. These findings have important implications in terms of maintaining long-term invertebrate biodiversity of mire sites.