Shotgun and TMT-Labeled Proteomic Analysis of the Ovarian Proteins of an Insect Vector,Aedes aegypti (Diptera: Culicidae)

Aedes aegypti [Linnaeus in Hasselquist; yellow fever mosquito] transmits several viruses that infect millions of people each year, including Zika, dengue, yellow fever, chikungunya, and West Nile. Pathogen transmission occurs during blood feeding. Only the females blood feed as they require a bloodmeal for oogenesis; in the bloodmeal, holo-transferrin and hemoglobin provide the females with a high iron load. We are interested in the effects of the bloodmeal on the expression of iron-associated proteins in oogenesis. Previous data showed that following digestion of a bloodmeal, ovarian iron concentrations doubles by 72 hr. We have used shotgun proteomics to identify proteins expressed in Ae. aegypti ovaries at two oogenesis developmental stages following blood feeding, and tandem mass tag-labeling proteomics to quantify proteins expressed at one stage following feeding of a controlled iron diet. Our findings provide the first report of mosquito ovarian protein expression in early and late oogenesis. We identify proteins differentially expressed in the two oogenesis development stages. We establish that metal-associated proteins play an important role in Ae. aegypti oogenesis and we identify new candidate proteins that might be involved in mosquito iron metabolism. Finally, this work identified a unique second ferritin light chain subunit, the first reported in any species. The shotgun proteomic data are available via ProteomeXchange with identifier PXD005893, while the tandem mass tag-labeled proteomic data are available with identifier PXD028242.     

Ellagitannin composition of blackberry as determined by HPLC-ESI-MS and MALDI-TOF-MS

Blackberries ( Rubus sp.) were evaluated by high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) to identify the ellagitannins present in flesh, torus (receptacle tissue), and seeds. Most ellagitannins were present (or detectable) only in seed tissues. Ellagitannins identified by HPLC-ESI-MS in the seeds included pedunculagin, casuarictin/potentillin, castalagin/vescalagin, lambertianin A/sanguiin H-6, lambertianin C, and lambertianin D. For several of the ellagitannins, isomeric separation was also obtained. The MALDI-TOF-MS analysis was primarily utilized to evaluate and identify high molecular mass (>1000 Da) ellagitannins. The MALDI analysis verified the presence of the ellagitannins identified by HPLC-ESI-MS including lambertianin A/sanguiin H-6, lambertianin C, and lambertianin D, but the analysis also indicated the presence of several other compounds that were most likely ellagitannins based on the patterns observed in the masses (i.e., loss or addition of a gallic acid moiety to a known ellagitannin). This study determined the presence of several possible isomeric forms of ellagitannins previously unidentified in fruit and presents a possible analytical HPLC method for the analysis of the major ellagitannins present in the fruit.     

Impact of fire regimes,logging and topography on hollows in fallen logs in eucalypt forest of south eastern Australia

Log hollows provide important habitat for a range of vertebrate fauna. Despite this, little is known about the impact fire regimes have on this resource, or the role topography may play in preserving hollows through the mitigation of fire intensity. This study examined the effect different combinations of fire frequency and topography have on (i) the number of hollow bearing logs and (ii) the presence and size of hollows within logs. The influence of wildfire severity and logging were also examined. Hollow availability (i.e. density of hollow bearing logs and hollow presence within logs) was greatest at sites burnt at ‘low’ frequency. The density of hollow logs was greater in gullies, though the effect of fire frequency did not vary with topographic position. Hollows showing signs of internal fire charring typically had a greater entrance width than unburnt hollows, which suggests fire plays an important role in creating large hollows. The number of hollow bearing logs increased with logging intensity, due to unmerchantable timber being left in situ. Wildfire severity had little effect on log hollows. The results indicate that frequent burning may reduce hollow availability, though it is likely that gullies will still retain a high density of hollow bearing logs irrespective of burning, and may play an important role in preserving connectivity of this resource across landscapes.     

Characterization of Anopheles gambiae (African Malaria Mosquito) Ferritin and the Effect of Iron on Intracellular Localization in Mosquito Cells

Ferritin is a 24-subunit molecule, made up of heavy chain (HC) and light chain (LC) subunits, which stores and controls the release of dietary iron in mammals, plants, and insects. In mosquitoes, dietary iron taken in a bloodmeal is stored inside ferritin. Our previous work has demonstrated the transport of dietary iron to the ovaries via ferritin during oogenesis. We evaluated the localization of ferritin subunits inside CCL-125 [Aedes aegypti Linnaeus (Diptera: Culicidae), yellow fever mosquito] and 4a3b [Anopheles gambiae Giles (Diptera: Culicidae), African malaria mosquito] cells under various iron treatment conditions to further elucidate the regulation of iron metabolism in these important disease vectors and to observe the dynamics of the intracellular ferritin subunits following iron administration. Deconvolution microscopy captured 3D fluorescent images of iron-treated mosquito cells to visualize the ferritin HC and LC homologue subunits (HCH and LCH, respectively) in multiple focal planes. Fluorescent probes were used to illuminate cell organelles (i.e., Golgi apparatus, lysosomes, and nuclei) while secondary probes for specific ferritin subunits demonstrated abundance and co-localization within organelles. These images will help to develop a model for the biochemical regulation of ferritin under conditions of iron exposure, and to advance novel hypotheses for the crucial role of iron in mosquito vectors.     

Incorporation of yolk fatty acids into body lipids of goldfish (Carassius auratus L.) larvae raised at two different temperatures

In five separate experiments, eggs from a single female goldfish were fertilized at 20°C. They were incubated at 22°C for 6 hours, after which some of the eggs were transferred to 13°C. When a defined post-hatch developmental stage was reached, lipid extracts were prepared from larvae, both with yolk sacs intact and after removal of the yolk sac by dissection. Other larvae were sampled at yolk exhaustion. Gas chromatographic analysis of fatty acid profiles revealed that larvae incorporated 16:0, 18:0, 20:4 (n–6) and 22:6 (n–3) into their tissues in proportions higher than those present in the eggs from which they were derived. At 22°C, these trends were particularly apparent at yolk exhaustion. At 13°C, proportions of polyunsaturated fatty acids in the bodies of newly hatched larvae were higher than those in the 22°C larval bodies. Monounsaturated fatty acids were preferentially depleted during development, especially in larvae from high quality eggs. No dependence of egg quality, as assessed by larval viability at 22°C, on total egg lipid mass or fatty acid composition was found. Larvae from the lowest quality eggs showed a reduced preference for incorporation of (n–3) polyunsaturated fatty acids into their tissues.     

Supplementation of Chelated Magnesium to Diets of the Pacific White Shrimp,Litopenaeus vannamei,Reared in Low‐salinity Waters of West Alabama

Shrimp farmers using inland low‐salinity waters in west Alabama have traditionally used agricultural fertilizers (K‐Mag®, muriate of potash) to raise pond water levels of potassium (K) and magnesium (Mg) to improve the rearing medium for Litopenaeus vannamei. Laboratory and farm trials were performed to investigate the potential of using dietary supplementation of Mg instead of costly agricultural fertilizers. A 5‐wk laboratory trial was devised to test four diets with varying levels of Mg supplementation (0, 0.15, 0.30, and 0.60% Mg chelate) using a magnesium chelate (MgC)–amino acid complex. Juvenile shrimp were stocked into artificial low‐salinity water (5 ppt) designed to contain low levels of Mg. A farm trial was also conducted to test the same diets under field conditions. Although both laboratory and on‐farm trials revealed a trend for increased growth using the diet with the highest Mg supplement, results were not significantly different. The use of magnesium chelates as dietary supplements at levels higher than the requirement level to enhance survival, growth, and osmoregulatory capacity of shrimp reared in inland low‐salinity well waters appeared to have limited potential. Until effective specialized diet formulations are produced, farmers should continue to supplement pond waters with fertilizers containing K and Mg.     

Contrasting climate velocity impacts in warm and cool locations show that effects of marine warming are worse in already warmer temperate waters

Species responses to climate change are often measured at broad spatiotemporal scales, which can miss the fine-scale changes that are most relevant to conservation and fisheries management. We develop a scaleable geostatistical approach to assess how juvenile and adult fish distributions have been shaped by changes in bottom temperature and dissolved oxygen over a recent decade of warming in the northeast Pacific. Across 38 demersal fishes, biomass trends were associated negatively with warming and positively with dissolved oxygen, but when trends in both biomass and climate were converted to velocities—the speed and direction a population would have to move to maintain consistent conditions—the effect of temperature change differed depending on local conditions. In the warmest locations, warming velocities were associated with negative biotic velocities for 19 of 69 species-maturity combinations, and yet were almost always associated with stable or positive biotic velocities in the coolest locations (64 of 69). These spatially consistent biomass declines (negative biotic velocities) in the warmest locations and increases in cooler locations suggest a redistribution of species with the potential for new ecological and fisheries interactions. After controlling for temperature, the more spatially consistent effects of dissolved oxygen were often negative, suggesting a mechanism other than hypoxia avoidance—potentially changes in primary production. Our approach identifies the species and locations that are most sensitive to observed changes in the environment at any scale, thus facilitating future vulnerability assessments.