Development changes of cuticular hydrocarbons in Chrysomya rufifacies larvae: potential for determining larval age

Age determination is the basis of determining the postmortem interval using necrophagous fly larvae. To explore the potential of using cuticular hydrocarbons for determining the ages of fly larvae, changes of cuticular hydrocarbons in developing larvae of Chrysomya rufifacies (Macquart) (Diptera: Calliphoridae) were investigated using gas chromatography with flame-ionization detection and gas chromatography-mass spectrometry. This study showed that the larvae produced cuticular hydrocarbons typical of insects. Most of the hydrocarbons identified were alkanes with the carbon chain length of 21-31, plus six kinds of alkenes. The hydrocarbon composition of the larvae correlated with age. The statistical results showed that simple peak ratios of n-C29 divided by another eight selected peaks increased significantly with age; their relationships with age could be modelled using exponential or power functions with R(2) close to or > 0.80. These results suggest that cuticular hydrocarbon composition is a useful indicator for determining the age of larval C. rufifacies, especially for post-feeding larvae, which are difficult to differentiate by morphology.     

Evidence for a novel cryoprotective protein from freeze-tolerant larvae of the goldenrod gall fly Eurosta solidaginis

Third-instar larvae of the goldenrod gall fly Eurosta solidaginis (Diptera: Tephritidae) from populations in northern North America transition from freeze-susceptible to freeze-tolerant just prior to the onset of winter. While studies have documented the accumulation of carbohydrate cryoprotectants during this transition, protein cryoprotectants common to other freeze-tolerant species have not been reported in the gall fly. Using larvae collected from a population in Madison County, NY, which changes from freeze-susceptible to freeze-tolerant in early October, we assayed for the presence of factors that could preserve the catalytic activity of the cold-labile enzyme, rabbit muscle lactate dehydrogenase. Freezing this enzyme with a heat-stable, hydrophilic fraction derived from homogenates of both freeze-tolerant larvae and those in the process of becoming freeze-tolerant preserved between 70% and 80% of this enzyme's activity. Neither a comparable solution of bovine serum albumin nor the naturally-occurring carbohydrates (glycerol, sorbitol, or trehalose) conferred this level of cryoprotection. The putative cryoprotective protein from gall fly larvae did not bind to a weak anion exchanger, implying that its character may be cationic.     

The effects of temperature and season on the O2 consumption of third-instar larvae of the goldenrod gall fly (Eurosta solidaginis)

Abstract. Third-instar larvae of the goldenrod gall fly ( Eurosta solidaginis Fitch) live inside ball galls on goldenrod plants from summer to the following spring.Because galls are highly exposed to the weather, larvae experience substantial variations in body temperature.This study documents the oxygen consumption of gall fly larvae with regard to the effects of ambient temperature, seasonal conditioning, and prior exposure to subzero temperature.The body mass of larvae doubles between the late summer and the autumn; it subsequently undergoes a modest decline by early winter.The O₂, consumption of field-acclimatized larvae increases with ambient temperature, especially between 0 and 10°C (Q₁₀= 2.6-3.4).The thermal sensitivity of metabolism declines at higher ambient temperatures, most notably during the autumn/early winter.After exposure to 15°C for 1 week, autumn and early winter larvae maintain much lower rates of O₂ consumption than do late summer specimens.Prior exposure to -5°C for 24 h did not influence the O₂ consumption of larvae.Low thermal sensitivity of O₂ consumption, especially at higher ambient temperatures, is an energy-sparing mechanism during seasonal inactivity.Indeed, the persistence of this metabolic pattern in larvae exposed to 15°C suggests that they have entered a state of diapause.     

Differences in cold tolerance,desiccation resistance,and cryoprotectant production between three populations of <Emphasis Type="Italic">Eurosta solidaginis</Emphasis> collected from different latitudes

Possible links between cold-tolerance and desiccation resistance were examined between larvae of the goldenrod gall fly collected from Michigan, southern Ohio, and Alabama locations as their host plant senesced. After acclimation to 5°C, Michigan-collected larvae were more cold-tolerant (25% survival after a 96 h exposure to −40°C) than larvae from Ohio (10% survival) and Alabama (0% survival). Increased cold-tolerance was partially linked to higher concentrations of the cryoprotectant glycerol (Michigan: 500 ± 30 mmol; Ohio: 270 ± 20; Alabama: 220 ± 20). Moreover, cryoprotectants may have functioned to reduce rates of overall and cuticular water loss for Michigan larvae, 0.10 ± 0.01 and 0.037 ± 0.003 μg mm⁻² h⁻¹, respectively, values that were 40-44% lower than those for Ohio and Alabama larvae and may represent a link between desiccation resistance and cold-tolerance. After acclimation to 20°C, Alabama-collected larvae had metabolic rates that were 40% lower than those from Ohio and Michigan that averaged 0.100 ± 0.006 μl of CO₂ produced g⁻¹ h⁻¹. The lower metabolic rate of Alabama-collected larvae at 20°C likely resulted in reduced respiratory transpiration that may represent a mechanism to maintain water balance at the higher overwintering temperatures they typically experience.     

Energy and water conservation in frozen vs. supercooled larvae of the goldenrod gall fly, Eurosta solidaginis (fitch) (Diptera: Tephritidae)

Insects that tolerate severe cold during winter may either supercool or tolerate ice forming within the tissues of the body. To compare the relative advantages of freezing and supercooling, we measured rates of CO(2) production and water loss in frozen and supercooled goldenrod gall fly larvae (Eurosta solidaginis). As an important first step, we measured the time required for ice content and metabolic rate to stabilize upon freezing. Ice content stabilized after only three hours of freezing at -5 degrees C, whereas CO(2) production required 12 hours to stabilize. Subsequent experiments found that freezing greatly reduced both water loss and metabolic rate. Comparisons of supercooled and frozen larvae at -5 degrees C indicated that CO(2) production fell 47% with freezing and water loss decreased 35%. As temperature decreased to -10 and -15 degrees C, CO(2) production fell exponentially and was no longer detectable at -20 degrees C with our measurement system. Our results demonstrate that freezing significantly reduces energy consumption during the winter and may therefore improve winter survival and spring fecundity. The advantages of freezing over supercooling would drive selection toward insect freeze tolerance and also toward higher supercooling points to increase the duration of freezing each winter.     

The distribution of risk and density-dependent mortality in the galls of Eurosta solidaginis, the goldenrod gall fly

ABSTRACT.

• 1 This paper explores the net effect of a suite of mortality factors on a sedentary prey, the larvae of the goldenrod gall fly, Eurosta solidaginis Fitch (Diptera: Tephritidae).
• 2 Mortality is caused by unknown factors early in larval development, two species of parasitoid wasp (Hymenoptera: Eurytomidae), an inquiline beetle larva (Coleoptera: Mordellidae), and during the winter months downy woodpeckers Picoides pubescens (L.).
• 3 Distribution of mortality among galls relative to prey (gall) distribution was measured and discussed with respect to the distribution of relative risk of predation.
• 4 Galls are by and large contagiously distributed among quadrats, and mortality is distributed in a comparable pattern to that of galls.
• 5 The pattern of mortality on Eurosta larvae is neither density-dependent nor aggregated independently of gall distribution. Persistence in the system is probably a result of a combination of other factors such as adult mortality and early larval death which may have intergenerational density-dependent effects, and the linkage of locally unstable sub-populations via migration.

     

杠柳萃取物对小菜蛾幼虫的毒杀活性研究

以杠柳Periploca sepium Bunge的根为材料进行甲醇超声波提取,粗提物以水／氯仿（1：1,v／v）萃取获得杠柳根氯仿萃取物。测定了萃取物对小菜蛾Plutella xyllostellaL．3龄幼虫的胃毒和触杀活性。实验结果显示,在50μg／头剂量处理下24h和48h后,氯仿萃取物对小菜蛾3龄幼虫的胃毒死亡率分别为82．22％和92．59％,显著高于触杀死亡率58．89％和68．15％。这一结果表明,杠柳根中含有对小菜蛾幼虫具有较强胃毒作用的活性成分。     

Diapause development in frozen larvae of the goldenrod gall fly, Eurosta solidaginis fitch (diptera: tephritidae)

Seasonal changes in metabolic rate and the potential for morphological development demonstrated that third-instar larvae of the goldenrod gall fly, Eurosta solidaginis Fitch, exhibit a distinct winter diapause. Metabolic rate (CO2 production) was significantly lower from 15 October to 9 February than in early autumn (9 September) and spring (1 March) samples. The induction of diapause coincided with the development of cold-hardening, maximum larval mass, and gall senescence, but our experiments did not identify specific cues triggering diapause induction. We examined the influence of exposure to 0 degrees C and -20 degrees C on diapause development. Diapause development in larvae stored at 0 degrees C occurred at approximately the same rate as in nature. Until 15 December the larvae were in the refractory phase of diapause (incapable of morphological development, even at permissive temperatures), but afterward moved to the activated phase within which diapause intensity decreased until termination in February. Diapause development occurred in larvae collected during the winter and stored at -20 degrees C for periods of 1 week to 3 months. Diapause intensity decreased in frozen larvae through the winter but at a slower rate than in larvae stored at 0 degrees C.     

Effect of mating stage on water balance, cuticular hydrocarbons and metabolism in the desert harvester ant, Pogonomyrmex barbatus

Water-loss rates increase after mating in queens of the harvester ant Pogonomyrmex barbatus (Formicidae: Myrmicinae), then increase again after the mated queens excavate an incipient nest. We determined the mechanistic basis for these increased water-loss rates by examining cuticular permeability, respiratory water loss, metabolic rates, and cuticular hydrocarbons for queens at three stages in the mating sequence: unmated alate queens, newly mated dealate queens, and mated queens excavated from their incipient nest. Both total water loss and cuticular transpiration increased significantly following mating, with cuticular transpiration accounting for 97% of the increased water loss. In contrast, metabolic rate and respiratory water loss were unaffected by mating stage. The total quantity of cuticular hydrocarbons did not vary by mating stage. However, relative amounts of four of the most abundant cuticular hydrocarbons did vary by mating stage, as did quantities of n-alkanes and methylalkanes. The general pattern was that percent composition of n-alkanes decreased through the mating sequence, while percent composition of methylalkanes increased over the same sequence. We discuss three mechanisms that might cause these post-mating increases in cuticular permeability. Our data support the hypothesis that part of this increase results from soil particles abrading the cuticle during the process of nest excavation.     

Cuticular pheromones and water balance in the house fly,Musca domestica

Epicuticular lipids serve two major roles in insects. Their waterproofing properties are crucial to survival in terrestrial environments, and they serve as contact pheromones in a wide array of taxa. Both functions may be affected by the physical properties of the surface lipids. This provides the opportunity for natural selection on water conservation, mediated by lipid phase behavior, to interact with and perhaps conflict with sexual selection on communication and mate recognition. We used the common house fly, Musca domestica, as a model for these interacting selective forces. Male house flies preferred female models treated with a high melting-point lipid mixture, suggesting that sexual and natural selection may both act to favor longer-chain, more saturated hydrocarbons. However, higher melting points did not result in lower rates of water loss. We propose a working model in which phase separation between the unsaturated female pheromone and saturated hydrocarbons results in areas of melted, pheromone-rich lipids and regional variation in cuticular permeability.     

Acquisition of freezing tolerance in early autumn and seasonal changes in gall water content influence inoculative freezing of gall fly larvae,Eurosta solidaginis (Diptera,Tephritidae)

We examined seasonal changes in freeze tolerance and the susceptibility of larvae of the gall fly, Eurosta solidaginis to inoculative freezing within the goldenrod gall (Solidago sp.). In late September, when the water content of the galls was high (approximately 55%), more than half of the larvae froze within their galls when held at -2.5 degrees C for 24 h, and nearly all larvae froze at -4 or -6 degrees C. At this time, most larvae survived freezing at > or = -4 degrees C. By October plants had senesced, and their water content had decreased to 33%. Correspondingly, the number of larvae that froze by inoculation at -4 and -6 degrees C also decreased, however the proportion of larvae that survived freezing increased markedly. Gall water content reached its lowest value (10%) in November, when few larvae froze during exposure to subzero temperatures > or = -6 degrees C. In winter, rain and melting snow transiently increased gall water content to values as high as 64% causing many larvae to freeze when exposed to temperatures as high as -4 degrees C. However, in the absence of precipitation, gall tissues dried and, as before, larvae were not likely to freeze by inoculation. Consequently, in nature larvae freeze earlier in the autumn and/or at higher temperatures than would be predicted based on the temperature of crystallization (T(c)) of isolated larvae. However, even in early September when environmental temperatures are relatively high, larvae exhibited limited levels of freezing tolerance sufficient to protect them if they did freeze.     

The use of non-aqueous chloroform/methanol extraction for the delipidation of brain with minimal loss of enzyme activities.

When freeze-dried brain was extracted at -4-0degrees C with dry chloroform/methanol (2:1 v/v), four of the five enzymes examined were recovered in the diethyl ether-washed residue without inactivation. By contrast, extraction with chloroform/methanol (2:1 v/v) in the presence of water destroyed activities of all the enzymes examined. The amounts of major lipids extracted were similar whether extraction was done in the absence or presence of water. The study was carried out with special interest in 2':3'-cyclic nucleotide 3'-phosphodiesterase (EC 3.1.4.37), which is firmly bound to the membrane structures of brain white matter.     

Water-Proofing Properties of Cuticular Lipids

SYNOPSIS. Epicuticular lipids play a critical role in allowingarthropods to thrive in terrestrial environments, by reducingtranspiration of water through the cuticle. These lipids consistof a diverse array of compounds, especiaUy long-chain hydrocarbons.Rates of water loss are correlated with hydrocarbon structuralfeatures, including chain length, unsaturation and methyl-branching.The water-proofing abilities of cuticular lipids appear to dependlargely on their physical properties. In most arthropods, ratesof water loss increase rapidly above a "transition" temperature.A widely accepted model proposes that this transition is dueto melting of the surface lipids to a fluid, permeable state.Evidence for this hypothesis has primarily been correlative,due to experimental limitations. Recent technical advances inlipid biophysics and water loss measurements have made it possibleto test the lipid melting model more directly. Experiments usingmodel cuticles, in vitro preparations and intact arthropodssupport the idea that the phase behavior of cuticular lipidsis a major factor determining cuticular permeability.     

Cuticular cavities: storage chambers for cyanoglucoside-containing defensive secretions in larvae of a Zygaenid moth

As a defensive reaction against predators the larvae of Zygaena trifolii Esper. 1783, release highly viscous fluid droplets out of cuticular cavities. The fluid appears on the cuticular surface upon contraction of the irritated segments, with no specialized muscles being involved. Two morphologically different types of cavities have been found: the larger ones are located beneath pigment spots, the smaller ones occupy the remaining surface except in the ventral region. Both types have complicated cuticular opening structures. The defensive fluid contains the cyanoglucosides linamarin and lotaustralin, the amino acid beta-cyano-L-alanine, proteins and water. Although a considerable amount of fluid (3-6 microliter per sixth-instar larva) is stored in the cuticle, fine structural examinations of the epidermis do not show any specific cells or cell areas with morphological adaptations for secretion. Further, there do not exist any major cytological differences between the cells below the cavities and in the ventral region, where those cavities are absent.     

Does R gene resistance allow wheat to prevent plant growth effects associated with Hessian fly (Diptera: Cecidomyiidae) attack?

Resistance genes (R genes) are an important part of the plant's immune system. Among insects, the Hessian fly, Mayetiola destructor (Say) (Diptera: Cecidomyiidae), larva is the target of the greatest number of characterized R genes (H1-H32). The biochemical/molecular mechanism of R gene resistance to Hessian fly is not well understood. In the absence of an effective R gene, larvae caused extensive growth deficits (> 30 cm) in wheat seedlings. In the presence of one of three effective R genes, H6, H9, or H13, larvae caused small growth deficits (approximately 3-4 cm) in two leaves (third and fourth) that were actively growing during the first days of larval attack. After larvae died on R gene plants, the fifth leaf and tiller leaves exhibited small increases in growth (2-4 cm). Growth responses of susceptible and resistant plants diverged at a time when Hessian fly larvae were establishing a nutritive gall tissue at feeding sites. The results of this study support the hypothesis that R gene resistance cannot prevent initial larval attack, but, by stopping the formation of the larval gall, it prevents the most serious consequences of larval attack.     

Food wrapping by females of the European Beewolf, Philanthus triangulum, retards water loss of larval provisions

Abstract.  Females of the European beewolf, Philanthus triangulum F. (Hymenoptera, Crabronidae), embalm the provisions of their larvae, paralyzed honeybees, with a secretion from a postpharyngeal gland prior to oviposition. This food wrapping is known to delay fungus infestation of the prey. In the present study, the hypothesis that the food wrapping has an additional function, namely the prevention of prey desiccation, is tested. Water loss of paralyzed but unembalmed honeybees and embalmed honeybees is measured and the composition and quantity of their cuticular hydrocarbons analyzed by coupled gas chromatography–mass spectrometry. Water loss is significantly lower in embalmed compared with unembalmed bees. This might have important advantages for the larvae under the warm and dry conditions that prevail in some brood cells. The embalming by beewolf females increases the total amount of hydrocarbons on the surface of the bees by almost ten-fold. Moreover, the proportion of unsaturated and short-chained hydrocarbons is significantly increased. Unsaturated and short-chain hydrocarbons are usually less effective against water loss, so the increased protection against water loss appears to be mediated mainly by the thickness of the hydrocarbon layer.     

Mechanisms to reduce dehydration stress in larvae of the Antarctic midge, Belgica antarctica

The Antarctic midge, Belgica antarctica, is exposed to frequent periods of dehydration during its prolonged larval development in the cold and dry Antarctic environment. In this study, we determined the water requirements of the larvae and the mechanisms it exploits to reduce the stress of drying. Larvae lost water at an exceptionally high rate (>10%/h) and tolerated losing a high portion (>70%) of their water content. Larvae were unable to absorb water from subsaturated water vapor (< or = 0.98 a(v)) to replenish their water stores, thus this midge relies exclusively on the intake of liquid water to increase its pool of body water and maintain water balance. To reduce dehydration stress, the midge employed a variety of mechanisms. Behaviorally, the larvae suppressed water loss by clustering. In response to slow dehydration, glycerol concentration increased 2-fold and trehalose concentration increased 3-fold, responses that are known to decrease the rate of water loss and increase dehydration tolerance. No changes in the mass of cuticular lipids occurred in response to desiccation, but the observed shift to longer hydrocarbons likely contributes to reduced water loss as the larvae dehydrate. As the larvae dehydrated, their oxygen consumption rate dropped, resulting in a reduction of water loss by respiration. Lastly, one bout of slow dehydration also enhanced the larva's ability to survive subsequent dehydration, suggesting that the larvae have the capacity for drought acclimation. Thus, these hydrophilic midge larvae prevent dehydration by multiple mechanisms that collectively reduce the water loss rate and increase dehydration tolerance.     

Nutrition as a facilitator of host‐race formation: the shift of a stem‐boring beetle to a gall host

1. The importance of host‐race formation to herbivorous insect diversity depends on the likelihood that successful populations can be established on a new plant host. A previously unexplored ecological aid to success on a novel host is better nutritional quality. The role of nutrition was examined in the shift of the stem‐boring beetle Mordellistena convicta to fly‐induced galls on goldenrod and the establishment there of a genetically distinct gall host race. 2. First, larvae of the host race inhabiting stems of Solidago gigantea were transplanted into stems and galls of greenhouse‐grown S. gigantea plants. At the end of larval development, the mean mass of larvae transplanted to galls was significantly greater than the mass of larvae transplanted to stems, indicating a likely nutritional benefit during the shift. This advantage was slightly but significantly diminished when the gall‐inducing fly feeding at the centre of the gall died early in the season. Additionally, there was a suggestion of a trade‐off in the increased mortality of smaller beetle larvae transplanted into galls. 3. In a companion experiment, S. gigantea gall‐race beetle larvae were likewise transplanted to S. gigantea stems and galls. Besides the expected greater mass in galls, the larvae also exhibited adaptations to the gall nutritional environment: larger inherent size, altered tunnelling behaviour, and no diminution of mass pursuant to gall‐inducer mortality. 4. In a third line of inquiry, chemical analyses of field‐collected S. gigantea plants revealed higher levels of mineral elements important to insect nutrition in galls as compared with stems.     

Delipidization of membrane glycoproteins solubilized in acidified chloroform/methanol. Preparation of N-retinylidene opsin in a water soluble form without-detergent

Bovine retina membrane proteins and glycoproteins were insoluble in chloroform/methanol (2:1, v/v) unless the membrane suspension was precipitated with trichloroacetic acid and the organic solvent mixture added to the precipitated membranes. The presence of millimolar amount of trichloroacetic acid in the organic solvent led to the total solubilization of membranes. The glycoproteins precipitated at the interphase after partition of the acidified chloroform/methanol solution with water and were resolubilized from the interphase with chloroform/methanol/water (1:1:0.3, by vol). The solubility properties of the membrane glycoproteins in the acidified organic solvent mixtures allow to remove the bulk of membrane lipids and to recover from the chloroform/methanol/water solution the glycoprotein of rod outer segment membranes, rhodopsin, as protonated N-retinylidene opsin in a water soluble form.     

Insect cytokine growth-blocking peptide may regulate density-dependent phase trait of cuticular melanization in the larval armyworm,Mythimna separata

Growth-blocking peptide (GBP) is a 25 amino acid insect cytokine found in lepidopteran insects that has diverse biological activities such as larval growth regulation, paralysis induction, cell proliferation and stimulation of immune cells. Density-dependent phase polyphenism is a phenomenon of phenotypic plasticity in which the expression of a variety of traits can be affected by local population density. In the present study, the armyworm Mythimna separata larvae with four rearing densities (1 larva/vial, 2 larvae/vial, 4 larvae/vial and 6 larvae/vial) were tested for cuticular melanization and body weight throughout the third-fifth instar, and the functional role of GBP in regulating the changes was investigated. The results indicated that when reared at high densities, the larvae exhibited less body weight and more degree of cuticular melanization than larvae reared at low densities. The gene expression of GBP in armyworm larvae showed an initial rise and then decline trend with increased rearing densities in the third to fifth instar. Compared with control, more degree of cuticular melanization was observed in GBP-injected larvae (500 ng/larva in volume 50 μL) than that in Ringer’s solution-injected counterparts. Furthermore, the gene expression level of dopa decarboxylase and prophenoloxidase increased significantly in GBP-injected fifth instar larvae from 6 h to 12 h after injection, suggesting the role of GBP in modulating density-dependent phase trait of armyworm cuticular melanization.