Isolation and characterization of hemolymph antifreeze proteins from larvae of the longhorn beetle Rhagium inquisitor (L.)

We describe a simple and effective procedure to isolate antifreeze proteins (AFPs) from the hemolymph of larvae of the longhorn beetle Rhagium inquisitor, and present some characteristics of their structures. Several AFPs were isolated from the hemolymph of this species by heat and acid extraction followed by cation exchange. The hemolymph contains at least six AFPs ranging in size from 12.5 to 12.8 kDa. Of these, three were separated to purity by the ion exchange step, as indicated by mass spectrometry. The remaining three forms were further separated by size exclusion chromatography, but could not be isolated to purity. All AFPs in the hemolymph of this species appears to have isoelectric points above 8.00. The dominant form, RiAFP(H4), was purified by the ion exchange step. Its amino acid composition reveals a lower level of cysteine and a higher level of threonine, arginine, alanine and glycine than seen in other insect AFPs. Its trypsin fingerprint does not match that of any known protein. It interacts with ice both in the anionic and cationic state.     

Low thermodynamic but high kinetic stability of an antifreeze protein from Rhagium mordax

The equilibrium heat stability and the kinetic heat tolerance of a recombinant antifreeze protein (AFP) from the beetle Rhagium mordax (RmAFP1) are studied through differential scanning calorimetry and circular dichroism spectroscopy. In contrast to other insect AFPs studied with this respect, the RmAFP1 has only one disulfide bridge. The melting temperature, T_m, of the protein is determined to be 28.5°C (pH 7.4), which is much lower than most of those reported for AFPs or globular proteins in general. Despite its low melting temperature, both biophysical and activity measurements show that the protein almost completely refolds into the native state after repeated exposure of 70°C. RmAFP1 thus appears to be kinetically stable even far above its melting temperature. Thermodynamically, the insect AFPs seem to be dividable in three groups, relating to their content of disulfide bridges and widths of the ice binding motifs; high melting temperature AFPs (high disulfide content, TxT motifs), low melting temperature but high refolding capability AFPs (one disulfide bridge, TxTxTxT motifs) and irreversibly unfolded AFPs at low temperatures (no disulfide bridges, TxTxTxTxT motifs). The property of being able to cope with high temperature exposures may appear peculiar for proteins which strictly have their effect at subzero temperatures. Different aspects of this are discussed.     

The role of endogenous antifreeze protein enhancers in the hemolymph thermal hysteresis activity of the beetle Dendroides canadensis

Antifreeze proteins (AFPs) lower the freezing point of water by a non-colligative mechanism, but do not lower the melting point, therefore producing a difference between the freezing and melting points termed thermal hysteresis. Thermal hysteresis activity (THA) of AFPs from overwintering larvae of the beetle Dendroides canadensis is dependent upon AFP concentration and the presence of enhancers of THA which may be either other proteins or low molecular mass enhancers. The purpose of this study was to determine the relative contributions of endogenous enhancers in winter D. canadensis hemolymph.Winter hemolymph collected over four successive winters (1997-1998 to 2000-2001) was tested. The first three of these winters were the warmest on record in this area, while December of the final year was the coldest on record. Protein and low molecular mass enhancers raised hemolymph THA 60-97% and 35-55%, respectively, based on hemolymph with peak THA for each year collected over the four successive winters. However, the hemolymph AFPs were not maximally enhanced since addition of the potent enhancer citrate (at non-physiologically high levels) resulted in large increases in THA. ¹³NMR showed that glycerol was the only low molecular mass solute present in sufficiently high concentrations in the hemolymph to function as an enhancer. Maximum THA appears to be ∼8.5 °C.     

IkB genes encoded in Cotesia plutellae bracovirus suppress an antiviral response and enhance baculovirus pathogenicity against the diamondback moth, Plutella xylostella

An endoparasitoid wasp, Cotesia plutellae, parasitizes larvae of the diamondback moth, Plutella xylostella, with its symbiotic polydnavirus, C. plutellae bracovirus (CpBV). This study analyzed the role of Inhibitor-kB (IkB)-like genes encoded in CpBV in suppressing host antiviral response. Identified eight CpBV-IkBs are scattered on different viral genome segments and showed high homologies with other bracoviral IkBs in their amino acid sequences. Compared to an insect ortholog (e.g., Cactus of Drosophila melanogaster), they possessed a shorter ankyrin repeat domain without any regulatory domains. The eight CpBV-IkBs are, however, different in their promoter components and expression patterns in the parasitized host. To test their inhibitory activity on host antiviral response, a midgut response of P. xylostella against baculovirus infection was used as a model reaction. When the larvae were orally fed the virus, they exhibited melanotic responses of midgut epithelium, which increased with baculovirus dose and incubation time. Parasitized larvae exhibited a significant reduction in the midgut melanotic response, compared to nonparasitized larvae. Micro-injection of each of the four CpBV genome segments containing CpBV-IkBs into the hemocoel of nonparasitized larvae showed the gene expressions of the encoded IkBs and suppressed the midgut melanotic response in response to the baculovirus treatment. When nonparasitized larvae were orally administered with a recombinant baculovirus containing CpBV-IkB, they showed a significant reduction in midgut melanotic response and an enhanced susceptibility to the baculovirus infectivity.     

Evolution of Hyperactive,Repetitive Antifreeze Proteins in Beetles

Some organisms that experience subzero temperatures, such as insects, fish, bacteria, and plants, synthesize antifreeze proteins (AFPs) that adsorb to surfaces of nascent ice crystals and inhibit their growth. Although some AFPs are globular and nonrepetitive, the majority are repetitive in both sequence and structure. In addition, they are frequently encoded by tandemly arrayed, multigene families. AFP isoforms from the mealworm beetle, Tenebrio molitor, are extremely potent and inhibit ice growth at temperatures below −5°C. They contain a 12-amino acid repeat with the sequence TCTxSxxCxxAx, each of which makes up one coil of the β-helix structure. TxT motifs are arrayed to form the ice-binding surface in all three known insect AFPs: the homologous AFPs from the two beetles, T. molitor and Dendroides canadensis, and the nonhomologous AFP from the spruce budworm, Choristoneura fumiferana. In this study, we have obtained the cDNA and genomic sequences of additional T. molitor isoforms. They show variation in the number of repeats (from 6 to 10) which can largely be explained by recombination at various TCT motifs. In addition, phylogenetic comparison of the AFPs from the two beetles suggests that gene loss and amplification may have occurred after the divergence of these species. In contrast to a previous study suggesting that T. molitor genes have undergone positive Darwinian selection (selection for heterogeneity), we propose that the higher than expected ratio of nonsynonymous-to-synonymous substitutions might result from selection for higher AT content in the third codon position. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users. [Reviewing Editor: Dr. John Oakeshott]     

Non-Jumping Take off Performance in Beetle Flight （Rhinoceros Beetle Trypoxylus dichotomus）

In recent decades, the take-off mechanisms of flying animals have received much attention in insect flight initiation. Most of previous works have focused on the jumping mechanism, which is the most common take-off mechanism found in flying animals. Here, we presented that the rhinoceros beetle, Trypoxylus dichotomus, takes offwithout jumping. In this study, we used 3-Dimensional （3D） high-speed video techniques to quantitatively analyze the wings and body kinematics during the initiation periods of flight. The details of the flapping angle, angle of attack of the wings and the roll, pitch and yaw angles of the body were investigated to understand the mechanism of take-off in T. dichotomus. The beetle took off gradually with a small velocity and small acceleration. The body kinematic analyses showed that the beetle exhibited stable take-off. To generate high lift force, the beetle modulated its hind wing to control the angle of attack; the angle of attack was large during the upstroke and small during the downstroke. The legs of beetle did not contract and strongly release like other insects. The hind wing could be con- sidered as a main source of lift for heavy beetle.     

Different effects of paternal trans-generational immune priming on survival and immunity in step and genetic offspring

Paternal trans-generational immune priming, whereby fathers provide immune protection to offspring, has been demonstrated in the red flour beetle Tribolium castaneum exposed to the insect pathogen Bacillus thuringiensis. It is currently unclear how such protection is transferred, as in contrast to mothers, fathers do not directly provide offspring with a large amount of substances. In addition to sperm, male flour beetles transfer seminal fluids in a spermatophore to females during copulation. Depending on whether paternal trans-generational immune priming is mediated by sperm or seminal fluids, it is expected to either affect only the genetic offspring of a male, or also their step offspring that are sired by another male. We therefore conducted a double-mating experiment and found that only the genetic offspring of an immune primed male show enhanced survival upon bacterial challenge, while phenoloxidase activity, an important insect immune trait, and the expression of the immune receptor PGRP were increased in all offspring. This indicates that information leading to enhanced survival upon pathogen exposure is transferred via sperm, and thus potentially constitutes an epigenetic effect, whereas substances transferred with the seminal fluid could have an additional influence on offspring immune traits and immunological alertness.     

Nemtaode-Vector Relationships in the Pine Wilt Disease System

Pinewood nematode, Bursaphelenchus xylophilus, is the causal agent of pine wilt disease in North America and Japan. Dispersal stage dauer larvae are transported to new host trees on the body surface and within the tracheal system of several beetle species. Worldwide, 21 species of Cerambycidae, 1 genus of Buprestidae, and 2 species of Curculionidae are known to carry pinewood nematode dauer larvae upon emerging from nematode-infested trees. Five species of cerambycids in the genus Monochamus are known to transmit dauer larvae to new host trees, four North American species and one Japanese species. Primary transmission to healthy trees occurs through beetle feeding wounds on young branches. Secondary transmission to stressed trees or recently cut logs occurs through Monochamus oviposition sites.     

Red oilseed rape? The potential for manipulation of petal colour in control strategies for the pollen beetle (Meligethes aeneus)

The pollen beetle (Meligethes aeneus) is a major pest of oilseed rape (Brassica napus) at the inflorescence stage and is well known to prefer colours called yellow by human observers over many other colours. While commercial cultivars of oilseed rape have yellow flowers, little is known about the potential to manipulate host plant location and reduce subsequent infestation by this pest through variation in flower colour. We investigated the responses of pollen beetles to flowers of a white-petalled oilseed rape variety that had been dyed different colours in semi-field arena and field experiments. Flowers dyed blue or red were less heavily infested than those dyed yellow or the white flowers, indicating that blue and red flowers were less attractive than yellow and white ones. This response was most likely due to differences in petal colour because olfactometer studies showed that beetle responses to the odours of the coloured treatments did not differ. The comparatively high infestation of untreated white flowers is interpreted as a consequence of their high UV reflectance; the presence of a UV receptor in M. aeneus is suggested, and its role in visually guided insect–plant interactions in this species described. The potential for manipulation of petal colour in control strategies for the pollen beetle is discussed.     

Characterization of an antifreeze protein from the polar diatom Fragilariopsis cylindrus and its relevance in sea ice

Antifreeze proteins (AFPs), characterized by their ability to separate the melting and growth temperatures of ice and to inhibit ice recrystallization, play an important role in cold adaptation of several polar and cold-tolerant organisms. Recently, a multigene family of AFP genes was found in the diatom Fragilariopsis cylindrus, a dominant species within polar sea ice assemblages. This study presents the AFP from F. cylindrus set in a molecular and crystallographic frame. Differential protein expression after exposure of the diatoms to environmentally relevant conditions underlined the importance of certain AFP isoforms in response to cold. Analyses of the recombinant AFP showed freezing point depression comparable to the activity of other moderate AFPs and further enhanced by salt (up to 0.9 °C in low salinity buffer, 2.5 °C at high salinity). However, unlike other moderate AFPs, its fastest growth direction is perpendicular to the c-axis. The protein also caused strong inhibition of recrystallization at concentrations of 1.2 and 0.12 μM at low and high salinity, respectively. Observations of crystal habit modifications and pitting activity suggested binding of AFPs to multiple faces of the ice crystals. Further analyses showed striations caused by AFPs, interpreted as inclusion in the ice. We suggest that the influence on ice microstructure is the main characteristic of these AFPs in sea ice.     

Formation of the insect head involves lateral contribution of the intercalary segment, which depends on Tc-labial function

The insect head is composed of several segments. During embryonic development, the segments fuse to form a rigid head capsule where obvious segmental boundaries are lacking. Hence, the assignment of regions of the insect head to specific segments is hampered, especially with respect to dorsal (vertex) and lateral (gena) parts. We show that upon Tribolium labial (Tc-lab) knock down, the intercalary segment is deleted but not transformed. Furthermore, we find that the intercalary segment contributes to lateral parts of the head cuticle in Tribolium. Based on several additional mutant and RNAi phenotypes that interfere with gnathal segment development, we show that these segments do not contribute to the dorsal head capsule apart from the dorsal ridge. Opposing the classical view but in line with findings in the vinegar fly Drosophila melanogaster and the milkweed bug Oncopeltus fasciatus, we propose a “bend and zipper” model for insect head capsule formation.     

Isolation and characterization of type I antifreeze proteins from cunner, Tautogolabrus adspersus, order Perciformes

Antifreeze proteins (AFPs) are produced by many species of teleost fish that inhabit potentially lethal ice-laden seawater and afford them protection from freezing. To date type I AFPs have been fully characterized in two teleost orders: Pleuronectiformes and Scorpaeniformes. In this study, we report the isolation and complete characterization of a type I AFP present in fish from a third order: cunner (Tautogolabrus adspersus), order Perciformes (family Labridae). This protein was purified from blood plasma and found to belong to what is now known as classical type I AFP with their small size (mass 4095.16 Da), alanine richness (> 57 mol%), high α-helicity (> 99%) with the ability to undergo reversible thermal denaturation, 11 amino acid (ThrX(10)) repeat regions within the primary structure, the capacity to impart a hexagonal bipyramidal shaping to ice crystals and the conservation of an ice-binding site found in many of the other type I AFPs. Partial de novo sequencing of the plasma AFP accounted for approximately half of the peptide mass. Sequencing of a combined liver and skin cDNA library indicated that the protein is produced without a signal sequence. In addition the translated product of the AFP cDNA suggests that it codes for the AFP isolated from plasma. These results further solidify the hypothesis that type I AFPs are multiphyletic in origin and suggest that they represent remarkable examples of convergent evolution within three orders of teleost fish.     

Divergent and conserved roles of extradenticle in body segmentation and appendage formation, respectively, in the cricket Gryllus bimaculatus

The cricket Gryllus bimaculatus is a typical hemimetabolous intermediate germ insect, in which the processes of segmentation and appendage formation differ from those in Drosophila, a holometabolous long germ insect. In order to compare their developmental mechanisms, we have focused on Gryllus orthologs of the Drosophila developmental regulatory genes and studied their functions. Here, we report a functional analysis of the Gryllus ortholog of extradenticle (Gb′exd) using embryonic and parental RNA interference (RNAi) techniques. We found the following: (1) RNAi suppression of Gb′exd results in the deletion or fusion of body segments. Especially the head was often very severely affected. This gap-like phenotype may be related to reduced expression of the gap genes hunchback and Krüppel in early RNAi germbands. (2) In the appendages, several segments (podomeres) were fused. (3) Head appendages including the antenna were transformed to a leg-like structure consisting of at least one proximal podomere as well as several tarsomeres. The defects in appendages are reminiscent of the phenotype caused by large exd clones in Drosophila antennal discs. These findings led us to the conclusion that (1) Gb′exd is required for segment patterning in the gnathal to abdominal region, acting in a gap gene-like manner in the anterior region. (2) Gb′exd plays important roles in formation of the appendages and the determination of their identities, acting as a regulatory switch that chooses between the fates of head appendages versus the appendage ground state. Although functions of Gb′exd in appendage patterning appear fundamentally conserved between Gryllus and Drosophila, its role in body segmentation may differ from that of Drosophila exd.     

The overwintering physiology of the emerald ash borer, Agrilus planipennis fairmaire (coleoptera: buprestidae)

Ability to survive cold is an important factor in determining northern range limits of insects. The emerald ash borer (Agrilus planipennis) is an invasive beetle introduced from Asia that is causing extensive damage to ash trees in North America, but little is known about its cold tolerance. Herein, the cold tolerance strategy and mechanisms involved in the cold tolerance of the emerald ash borer were investigated, and seasonal changes in these mechanisms monitored. The majority of emerald ash borers survive winter as freeze-intolerant prepupae. In winter, A. planipennis prepupae have low supercooling points (∼−30 °C), which they achieve by accumulating high concentrations of glycerol (∼4 M) in their body fluids and by the synthesis of antifreeze agents. Cuticular waxes reduce inoculation from external ice. This is the first comprehensive study of seasonal changes in cold tolerance in a buprestid beetle.     

Modeling Pseudomonas syringae ice-nucleation protein as a beta-helical protein

Antifreeze proteins (AFPs) inhibit the growth of ice, whereas ice-nucleation proteins (INPs) promote its formation. Although the structures of several AFPs are known, the structure of INP has been modeled thus far because of the difficulty in determining membrane protein structures. Here, we present a novel model of an INP structure from Pseudomonas syringae based on comparison with two newly determined insect AFP structures. The results suggest that both this class of AFPs and INPs may have a similar beta-helical fold and that they could interact with water through the repetitive TXT motif. By theoretical arguments, we show that the distinguishing feature between an ice inhibitor and an ice nucleator lies in the size of the ice-interacting surface. For INPs, the larger surface area acts as a template that is larger than the critical ice embryo surface area required for growth. In contrast, AFPs are small enough so that they bind to ice and inhibit further growth without acting as a nucleator.     

AFP-Pred: A random forest approach for predicting antifreeze proteins from sequence-derived properties

Some creatures living in extremely low temperatures can produce some special materials called “antifreeze proteins” (AFPs), which can prevent the cell and body fluids from freezing. AFPs are present in vertebrates, invertebrates, plants, bacteria, fungi, etc. Although AFPs have a common function, they show a high degree of diversity in sequences and structures. Therefore, sequence similarity based search methods often fails to predict AFPs from sequence databases. In this work, we report a random forest approach “AFP-Pred” for the prediction of antifreeze proteins from protein sequence. AFP-Pred was trained on the dataset containing 300 AFPs and 300 non-AFPs and tested on the dataset containing 181 AFPs and 9193 non-AFPs. AFP-Pred achieved 81.33% accuracy from training and 83.38% from testing. The performance of AFP-Pred was compared with BLAST and HMM. High prediction accuracy and successful of prediction of hypothetical proteins suggests that AFP-Pred can be a useful approach to identify antifreeze proteins from sequence information, irrespective of their sequence similarity.     

Predation by Pterostichus melanarius (Illiger) (Coleoptera: Carabidae) on immature Rhagoletis mendax Curran (Diptera: Tephritidae) in semi-field and field conditions

Practices that enhance abundance and diversity of generalist predators are often employed with the objective of improving biological control of insect pests. Ground beetles and other predators can prey on blueberry maggot, an important pest of blueberries, when mature larvae pupate in the ground. We conducted mesocosm and field experiments to determine if Pterostichus melanarius, a common predatory ground beetle, lowers maggot numbers in compost mulch or when predator and alternative prey abundances are manipulated. At background (field) densities of alternative prey, increasing densities of P. melanarius did not significantly reduce pest numbers in mesocosms containing compost or soil. When alternative prey were removed from compost, beetles reduced pest numbers by up to 35%. In field experiments, maggot numbers were higher when beetles and other predators were excluded from soil plots, but beetle exclusion had no effect in compost plots where both predator and alternative prey numbers were high. Our results indicate that there can be some reduction of blueberry maggot by P. melanarius and other potential predators when there are few alternative prey. However, despite attracting large numbers of predators compost mulch did not lead to a significant reduction in blueberry maggot; in fact, the high abundance of alternative food associated with compost appeared to interfere with beetle predation on blueberry maggot.     

The mitochondrial genome of the Russian wheat aphid Diuraphis noxia: Large repetitive sequences between trnE and trnF in aphids

To characterize aphid mitochondrial genome (mitogenome) features, we sequenced the complete mitogenome of the Russian wheat aphid, Diuraphis noxia. The 15,784-bp mitogenome with a high A + T content (84.76%) and strong C skew (− 0.26) was arranged in the same gene order as that of the ancestral insect. Unlike typical insect mitogenomes, D. noxia possessed a large tandem repeat region (644 bp) located between trnE and trnF. Sequencing partial mitogenome of the cotton aphid (Aphis gossypii) further confirmed the presence of the large repeat region in aphids, but with different repeat length and copy number. Another motif (58 bp) tandemly repeated 2.3 times in the control region of D. noxia. All repeat units in D. noxia could be folded into stem-loop secondary structures, which could further promote an increase in copy numbers. Characterization of the D. noxia mitogenome revealed distinct mitogenome architectures, thus advancing our understanding of insect mitogenomic diversities and evolution.     

Sampling beetle communities: Trap design interacts with weather and species traits to bias capture rates

Globally, many insect populations are declining, prompting calls for action. Yet these findings have also prompted discussion about sampling methods and interpretation of long‐term datasets. As insect monitoring and research efforts increase, it is critical to quantify the effectiveness of sampling methods. This is especially true if sampling biases of different methods covary with climate, which is also changing over time. We assess the effectiveness of two types of flight intercept traps commonly used for beetles, a diverse insect group responsible for numerous ecosystem services, under different climatic conditions in Norwegian boreal forest. One of these trap designs includes a device to prevent rainwater from entering the collection vial, diluting preservatives and flushing out beetles. This design is compared to a standard trap. We ask how beetle capture rates vary between these traps, and how these differences vary based on precipitation levels and beetle body size, an important species trait. Bayesian mixed models reveal that the standard and modified traps differ in their beetle capture rates, but that the magnitude and direction of these differences change with precipitation levels and beetle body size. At low rainfall levels, standard traps catch more beetles, but as precipitation increases the catch rates of modified traps overtake those of standard traps. This effect is most pronounced for large‐bodied beetles. Sampling methods are known to differ in their effectiveness. Here, we present evidence for a less well‐known but likely common phenomenon—an interaction between climate and sampling, such that relative effectiveness of trap types for beetle sampling differs depending on precipitation levels and species traits. This highlights a challenge for long‐term monitoring programs, where both climate and insect populations are changing. Sampling methods should be sought that eliminate climate interactions, any biases should be quantified, and all insect datasets should include detailed methodological metadata.