Synthesis and biological activity of FGLamide allatostatin analogs with Phe3 residue modifications

A FGLamide allatostatin neuropeptide mimic ( H17 ) is a potential insect growth regulator which inhibits the production of juvenile hormone by the corpora allata. To find more evidence to reveal the structure–activity relationships of the Phe³ residue in the C‐terminal conserved pentapeptide and search for novel analogs with high activity, a series of Phe³ residue‐modified analogs were designed and synthesized using H17 as the lead compound. Bioassay using juvenile hormone (JH) production by corpora allata of the cockroach Diploptera punctata indicated that analogs 4 , 11 , and 13 showed strong ability to inhibit JH production in vitro, with IC₅₀ of 38.5, 22.5, and 26 nM, respectively. As well, the activity of analog 2 (IC₅₀: 89.5 nM) proved roughly equivalent to that of H17 . Based on the primary structure–activity relationships of Phe³ residue, we suggest that for analogs containing six‐membered aromatic rings, removing the methylene group of Phe³ or an o‐halogen or p‐halogen‐substituted benzene ring could increase the ability to inhibit biosynthesis of JH. This study will be useful for the design of new allatostatin analogs for insect management. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Comparative analysis of the methods used for finding surface energy to investigate protein interaction behavior on chromatographic supports

Hydrophobic interaction chromatography, an important and effective purification strategy, is generally used for the purification of variety of biomolecules. A basic understanding of the protein interaction behavior is required to effectively separate these biomolecules. A colloidal type extended Derjaguin, Landau, Verwey, and Overbeek calculations were utilized to study the interactions behavior of model proteins to commercially available hydrophobic chromatographic materials that is, Toyopearl Phenyl 650C and Toyopearl Butyl 650C. Physicochemical properties of selected model proteins were achieved by contact angle and zeta potential measurements. The contact angle of chromatographic materials used was achieved through sessile drop method on disrupted beads and capillary penetration method (CPM) on intact beads. The surface properties were further used to calculate the interactions of the proteins to chromatographic supports. The calculated secondary energy minimum of the proteins with the chromatographic materials (from the contact angle values determined through both methods can be correlated with the retention volumes from the real chromatography. The secondary energy minimum values are higher for each protein to the chromatographic materials calculated from the inputs derived through sessile drop method compared to CPM. For instance, immunoglobulin G has secondary energy minimum value of 0.17 kT compared to 0.11 kT, obtained through sessile drop method and CPM, respectively. Average relative values of the energy minimum calculated for all proteins are as 1.51 kT and 1.29 kT for Toyopearl Butyl 650C and Toyopearl Phenyl 650C, respectively, as a conversion factor for estimation of secondary energy minimum for both methods.     

Sources of variation in larval parasitism of two sympatrically outbreaking birch forest defoliators

1. Studies of insect communities rarely support the parasitoid–host regulation hypothesis. Spatio‐temporal variation in parasitoid prevalence due to complex food web interactions or abiotic factors may prevent parasitoids from regulating hosts. 2. We examined the relative contribution of spatial (altitude) and temporal (years) sources to total variation in parasitoid prevalence rates in outbreaks of Epirrita autumnata Borkhausen and Operophtera brumata Linnaeus populations. We tested whether prevalence rates of generalist parasitoids were correlated between sympatric host populations and to what extent any of the parasitoids were host density dependent. 3. Four larval parasitoids (two specialists and two generalists) exhibited significantly structured spatio‐temporal dynamics over years and altitudes. The prevalence rates of one of the generalists were spatio‐temporally correlated between the two host species, while for the other they were not. 4. Three parasitoids showed tendencies for direct or delayed positive density dependence as expected from numerical and functional responses to their hosts. However, the effects were weak and minute compared to the variation attributed to year and altitude. 5. We conclude that unknown aspects of the larval parasitoid ecology that co‐vary with altitude and year in the study system dominate their prevalence dynamics and thus act to hinder density‐dependent responses that could potentially regulate host populations.     

The Erwin equation of biodiversity: From little steps to quantum leaps in the discovery of tropical insect diversity

Almost 40 years ago, Terry L. Erwin published a seemingly audacious proposition: There may be as many as 30 million species of insects in the world. Here, we translate Erwin's verbal argument into a diversity-ratio model—the Erwin Equation of Biodiversity—and discuss how it has inspired other biodiversity estimates. We categorize, describe the assumptions for, and summarize the most commonly used methods for calculating estimates of global biodiversity. Subsequent diversity-ratio extrapolations have incorporated parameters representing empirical insect specialization ratios, and how insect specialization changes at different spatial scales. Other approaches include macroecological diversity models and diversity curves. For many insect groups with poorly known taxonomies, diversity estimates are based on the opinions of taxonomic experts. We illustrate our current understanding of insect diversity by focusing on the six most speciose insect orders worldwide. For each order, we compiled estimates of the (a) maximum estimated number of species, (b) minimum estimated number of species, and (c) number of currently described species. By integrating these approaches and considering new information, we believe an estimate of 5.5 million species of insects in the world is much too low. New molecular methodologies (e.g., metabarcoding and NGS studies) are revealing daunting numbers of cryptic and previously undescribed species, at the same time increasing our precision but also uncertainty about present estimates. Not until technologies advance and sampling become more comprehensive, especially of tropical biotas, will we be able to make robust estimates of the total number of insect species on Earth.     

Chromatin condensation and differential sensitivity of mammalian and insect cells to DNA strand breaks induced by bleomycin

Bleomycin (BLM) induces DNA damage in living cells. In this report we analyzed the role of chromatin compactness in the differential response of mosquito (ATC-15) and mammalian (CHO) cells to DNA strand breaks induced by BLM. We used cells unexposed and exposed to sodium butyrate (NaB), which induces chromatin decondensation. By nucleoid sedimentation assay and digestions of nuclei with DNAse I, untreated mosquito cells (no BLM; no NaB) were shown to have more chromatin condensation than untreated CHO cells. By alkaline unwinding ATC-15 cells treated with NaB showed more BLM-induced DNA strand breaks than NaB-untreated CHO cells. The time-course of BLM-induced DNA damage to nuclear DNA was similar for NaB-untreated mammalian and insect cells, but with mosquito cells showing less DNA strand breaks, both at physiological temperatures and at 4 °C. However, when DNA repair was inhibited by low temperatures and chromatin was decondensed by NaB treatments, differences in BLM-induced DNA damage between these cells lines were no longer observed. In both cell lines, NaB did not affect BLM action on cell growth and viability. On the other hand, the low sensitivity of ATC-15 cells to BLM was reflected in their better growth efficiency. These cells exhibited a satisfactory growth at BLM doses that produced a permanent arrest of growth in CHO cells. The data suggest that mosquito cells might have linker DNAs shorter than those of mammalian cells, which would result in the observed both greater chromatin condensation and greater resistance to DNA damage induced by BLM as compared to CHO cells.     

Direct peptide profiling of lateral cell groups of the antennal lobes of Manduca sexta reveals specific composition and changes in neuropeptide expression during development

The paired antennal lobes are the first integration centers for odor information in the insect brain. In the sphinx moth Manduca sexta, like in other holometabolous insects, they are formed during metamorphosis. To further understand mechanisms involved in the formation of this particularly well investigated brain area, we performed a direct peptide profiling of a well defined cell group (the lateral cell group) of the antennal lobe throughout development by MALDI-TOF mass spectrometry. Although the majority of the about 100 obtained ion signals represent still unknown substances, this first peptidomic characterization of this cell group indicated the occurrence of 12 structurally known neuropeptides. Among these peptides are helicostatin 1, cydiastatins 2, 3, and 4, M. sexta-allatotropin (Mas-AT), M. sexta-FLRFamide (Mas-FLRFamide) I, II, and III, nonblocked Mas-FLRFamide I, and M. sexta-myoinhibitory peptides (Mas-MIPs) III, V, and VI. The identity of two of the allatostatins (cydiastatins 3 and 4) and Mas-AT were confirmed by tandem mass spectrometry (MALDI-TOF/TOF). During development of the antennal lobe, number and frequency of ion signals including those representing known peptides generally increased at the onset of glomeruli formation at pupal Stage P7/8, with cydiastatin 2, helicostatin 1, and Mas-MIP V being the exceptions. Cydiastatin 2 showed transient occurrence mainly during the period of glomerulus formation, helicostatin 1 was restricted to late pupae and adults, while Mas-MIP V occurred exclusively in adult antennal lobes. The power of the applied direct mass spectrometric profiling lies in the possibility of chemically identifying neuropeptides of a given cell population in a fast and reliable manner, at any developmental stage in single specimens. The identification of neuropeptides in the antennal lobes now allows to specifically address the function of these signaling molecules during the formation of the antennal lobe network.     

Characterization of bimodal cell death of insect cells in a rotating‐wall vessel and shaker flask

In previous publications, we reported the benefits of a high‐aspect rotating‐wall vessel (HARV) over conventional bioreactors for insect‐cell cultivation in terms of reduced medium requirements and enhanced longevity. To more fully understand the effects that HARV cultivation has on longevity, the present study characterizes the mode and kinetics of Spodoptera frugiperda cell death in this quiescent environment relative to a shaker‐flask control. Data from flow cytometry and fluorescence microscopy show a greater accumulation of apoptotic cells in the HARV culture, by a factor of at least 2 at the end of the cultivation period. We present a kinetic model of growth and bimodal cell death. The model is unique for including both apoptosis and necrosis, and further, transition steps within the two pathways. Kinetic constants reveal that total cell death is reduced in the HARV and the accumulation of apoptotic cells in this vessel results from reduced depletion by lysis and secondary necrosis. The ratio of early apoptotic to necrotic cell formation is found independent of cultivation conditions. In the model, apoptosis is only well represented by an integral term, which may indicate its dependence on accumulation of some factor over time; in contrast, necrosis is adequately represented with a first‐order term. Cell‐cycle analysis shows the percent of tetraploid cells gradually decreases during cultivation in both vessels. For example, between 90% and 70% viability, tetraploid cells in the HARV drop from 43 ± 1% to 24 ± 4%. The data suggests the tetraploid phase as the likely origin for apoptosis in our cultures. Possible mechanisms for these changes in bimodal cell death are discussed, including hydrodynamic forces, cell–cell interactions, waste accumulation, and mass transport. These studies may benefit insect‐cell cultivation by increasing our understanding of cell death in culture and providing a means for further enhancing culture longevity. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 14–26, 1999.     

Ontogeny of tracheal system structure: a light and electron-microscopy study of the metathoracic femur of the American locust, Schistocerca americana

Does oxygen delivery become more challenging for insects as they increase in size? To partially test this hypothesis, we used quantitative light and electron microscopy to estimate the oxygen delivery capacity for two steps of tracheal oxygen delivery within the metathoracic femur (jumping leg) for 2nd instar (about 47 mg) and adult (about 1.7 g) locusts, Schistocerca americana. The fractional cross‐sectional areas of the major tracheae running longitudinally along the leg were similar in adults and 2nd instars; however, since the legs of adults are longer, the mass‐specific diffusive conductances of these tracheae were 4‐fold greater in 2nd instars. Diffusive gas exchange longitudinally along the leg is easily possible for 2nd instars but not adults, who have many air sacs within the femur. Mitochondrial content fell proximally to distally within the femur in 2nd instars but not adults, supporting the hypothesis that diffusion was more important for the former. Lateral diffusing capacities of the tracheal walls were 12‐fold greater in adults than 2nd instars. This was primarily due to differences in the smallest tracheal class (tracheoles), which had thinner epidermal and cuticular layers, greater surface to volume ratios, and greater mass‐specific surface areas in adults. Adults also had greater mitochondrial contents, larger cell sizes and more intracellular tracheae. Thus, larger insects do not necessarily face greater problems with oxygen delivery; adult grasshoppers have superior oxygen delivery systems and greater mass‐specific aerobic capacities in their legs than smaller/younger insects. J. Morphol. 262:800–812, 2004. © 2004 Wiley‐Liss, Inc.