Biological differences between strains of Tribolium castaneum selected for resistance to hypoxia and hypercarbia, and the unselected strain

Abstract. A strain of the red flour beetle Tribolium castaneum (Herbst) was selected separately for resistance to hypoxia and hypercarbia. At the 21st generation, adults and pupae of the line selected for resistance to hypoxia were significantly heavier than those selected for resistance to hypercarbia and the unselected line. It also exhibited a more prolonged developmental range than the unselected line. The line selected for resistance to hypercarbia was also significantly heavier than the unselected line. When the selected and unselected lines were subjected to starvation at the 26th generation, metabolism in both selected lines proceeded at a lower rate than in the unselected line and consequently the selected lines survived for much longer periods.     

Efficacy of Beauveria bassiana for control of Tribolium castaneum with reduced oxygen and increased carbon dioxide

This study investigated the effect of atmosphere modification, a widely adopted means of insect control in stored products, on the efficacy of Beauveria bassiana for one of the most difficult to control pests, Tribolium castaneum. Oxygen reduction to 5% (±1%) as opposed to CO₂ elevation to 40% (±2%) for the first 72 h of fungus exposure resulted in significantly greater larval mortality than fungus exposure under ambient atmospheres. Both treatments reduced pupation of older larvae suggesting that slowed development may be a beneficial factor for fungal efficacy. CO₂ elevation but not O₂ reduction significantly affected the mortality of adult beetles that were exposed to the fungus. Carbon dioxide elevation significantly reduced B. bassiana’s germination and growth rates, but oxygen reduction did not.     

赤拟谷盗的磷化氢敏感和抗性品系体内羧酸酯酶的活性比较

本文比较测定了赤拟谷盗Tribolium castaneum(Herbst)的磷化氢抗性(Rf=327)和敏感品系害虫的羧酸酯酶活性,研究了该害虫同一品系不同个体间羧酸酯酶的活性差异,比较了两品系害虫在系列磷化氢浓度下熏蒸24h和6.94×10-2mg/L磷化氢浓度下熏蒸不同时间的羧酸酯酶活性。主要结果为:未熏蒸的抗性害虫幼虫和蛹体内的羧酸酯酶活性分别高于敏感品系的1.37和1.16倍;敏感和抗性害虫同品系内不同个体间羧酸酯酶活性分布频率都存在明显差异,抗性害虫中酶活性大的个体数量占的比例较大;磷化氢浓度分别为0.69×10-2、2.78×10-2、5.56×10-2、8.33×10-2和11.11×10-2mg/L时都可导致敏感害虫羧酸酯酶的活性降低,但活性受抑制的程度不因浓度高低呈相应的增减。浓度分别为5.56×10-2、11.11×10-2、13.89×10-2、20.83×10-2和27.78×10-2mg/L的熏蒸中抗性害虫体内酶活性增加,且活性增高的程度与浓度增减也不呈对应变化。在6.94×10-2mg/L磷化氢浓度下熏蒸不同时间的结果中,敏感害虫的酶活性随时间延长而下降,抗性害虫的活性则随时间延长而增大。研究表明赤拟谷盗对磷化氢的抗性可能与羧酸酯酶的活性增加有关。     

Effects of assortative mating for pupa weight on genetic parameters of unselected Tribolium castaneum

Summary Effects of random (R) or positive assortative (A) mating for pupal weight (PW) on genetic parameters of pupation time (PT), pupal and larval weights (LW) were studied in unselected populations of Tribolium castaneum. Two groups, each with 50 males mated to 100 females in each of 5 replicates, were either R-mated or A-mated for 3 generations. Genetic parameters were estimated from covariances between sibs (R group) or by an iterative method (A group). Estimates of heritability in R and A groups were 0.30±0.12 and 0.39±0.02 (PW); 0.26±0.13 and 0.49±0.04 (LW); and 0.39±0.10 and 0.25±0.03 (PT). Estimates of genetic correlations in the R group were –0.21±0.23 (PW and LW); 0.45±0.10 (PW and PT); and –0.77±0.14 (LW and PT). Those in the A group were 0.27±0.10 (PW and LW); 0.15±0.14 (PW and PT); the genetic correlation between LW and PT was not estimable in this group. Within-family variances (grams squared) of PW by generation (1, 2, and 3) were, respectively: 0.048 (R) and 0.047 (A); 0.054 (R) and 0.041 (A); and 0.050 (R) and 0.046 (A). In agreement with theory, estimates of heritability of PW and LW were larger in the A group. Estimates of genetic correlations in the A group were inconsistent with expectations from theory. Assortative mating tended to decrease within-family variance of PW.     

Inheritance pattern and expression of resistance to phosphine in larval stage of Tribolium castaneum (Coleoptera: Tenebrionidae)

Resistance developed by Tribolium castaneum (Herbst), a major pest of stored grain and grain products, to phosphine is a serious concern. Treatment of phosphine is given to all the stages (egg, larva, pupa and adult) of insects simultaneously in the infected commodity. Effective resistance management tactics need a proper understanding of expression of resistance in relation to dosage in immature stages including larvae. So, this study was undertaken to understand the genetic basis of phosphine resistance in larval stage of T. castaneum by crossing the resistant and susceptible parents to obtain the F₁, F₂ and the back-cross progenies. The resistant parent had a resistance ratio of 37.75 when compared to the susceptible parent. The susceptibility levels of the reciprocal F₁ hybrids did not show any significant difference indicating autosomal mode of inheritance of resistance. Level of dominance for the reciprocal F₁ hybrids was estimated (-0.70 and ?0.68) which showed that the resistance is expressed as incompletely recessive in larval stage of T. castaneum. χ² analysis showed that the observed and expected mortalities differed significantly (P < 0.05) in most of the concentrations indicating that more than one gene is responsible for phosphine resistance. Two plateaus at ～ 40 % and ～ 90 % mortality levels for the back-cross progeny to the resistant parent further confirmed the involvement of two or more genes in imparting resistance to phosphine. Our results will aid in enhancing the knowledge on the development of resistance to phosphine in the field and thus facilitate in designing suitable resistance management tactics.     

Larval RNA Interference in the Red Flour Beetle,Tribolium castaneum

The red flour beetle, Tribolium castaneum, offers a repertoire of experimental tools for genetic and developmental studies, including a fully annotated genome sequence, transposon-based transgenesis, and effective RNA interference (RNAi). Among these advantages, RNAi-based gene knockdown techniques are at the core of Tribolium research. T. castaneum show a robust systemic RNAi response, making it possible to perform RNAi at any life stage by simply injecting double-stranded RNA (dsRNA) into the beetle’s body cavity.In this report, we provide an overview of our larval RNAi technique in T. castaneum. The protocol includes (i) isolation of the proper stage of T. castaneum larvae for injection, (ii) preparation for the injection setting, and (iii) dsRNA injection. Larval RNAi is a simple, but powerful technique that provides us with quick access to loss-of-function phenotypes, including multiple gene knockdown phenotypes as well as a series of hypomorphic phenotypes. Since virtually all T. castaneum tissues are susceptible to extracellular dsRNA, the larval RNAi technique allows researchers to study a wide variety of tissues in diverse contexts, including the genetic basis of organismal responses to the outside environment. In addition, the simplicity of this technique stimulates more student involvement in research, making T. castaneum an ideal genetic system for use in a classroom setting.     

营养质量对雄性赤拟谷盗生长发育、抗饥饿和资源再获取能力的影响

昆虫的生长发育、抗饥饿和资源再获取能力对其生存、生殖和性选择非常重要。本文研究了营养质量对赤拟谷盗Tribolium castaneum(Herbst)生长发育、抗饥饿和资源再获取能力的影响。结果表明,三个营养处理下雄性赤拟谷盗的生长发育性状（幼虫重、发育历期、蛹重和成虫重）均存在显著性差异,在高营养质量下生长的雄性赤拟谷盗发育更快,体重更大,且各生长发育性状间存在显著性相关。营养质量对雄性赤拟谷盗成虫的抗饥饿能力没有显著影响,但对其资源再获取能力有显著影响,表现为在高营养质量下生长的雄性赤拟谷盗成虫比在低营养质量下生长的具有更高的资源再获取能力。雄性赤拟谷盗抗饥饿能力和资源再获取能力之间存在显著性相关,但是雄性赤拟谷盗抗饥饿能力和资源再获取能力除了与发育历期存在显著性相关外,与其它生长发育性状不存在显著性相关。本文还对这些实验结果的性选择意义进行了讨论。     

Strain-specific priming of resistance in the red flour beetle, Tribolium castaneum

As invertebrates lack the molecular machinery employed by the vertebrate adaptive immune system, it was thought that they consequently lack the ability to produce lasting and specific immunity. However, in recent years, it has been demonstrated that the immune defence of invertebrates is by far more complicated and specific than previously envisioned. Lasting immunity following an initial exposure that proves protection on a secondary exposure has been shown in several species of invertebrates. This phenomenon has become known as immune priming. In the cases where it is explicitly tested, this priming can also be highly specific. In this study, we used survival assays to test for specific priming of resistance in the red flour beetle, Tribolium castaneum, using bacteria of different degrees of relatedness. Our results suggest an unexpected degree of specificity that even allows for differentiation between different strains of the same bacterium. However, our findings also demonstrate that specific priming of resistance in insects may not be ubiquitous across all bacteria.     

Identification of microsatellite markers in the red flour beetle,Tribolium castaneum

We isolated and characterized microsatellite markers for the red flour beetle Tribolium castaneum, an important model species for studies in various areas of evolutionary biology and ecology. A microsatellite‐enriched genomic library was constructed and screened with single stranded oligonucleotide probes [(CCT)₁₇, (AAT)₁₇ and (CAG)₁₇]. Forty‐five primer pairs were designed of which 19 pairs produced successful amplification. Polymorphism screening involved beetles from five beetle strains and revealed 15 polymorphic and four monomorphic markers. The development of polymorphic microsatellite markers will facilitate future ecological and genetic studies involving T. castaneum beetles.     

Tribolium confusum responses to blends of cereal kernels and plant volatiles

In this study, we report on the behavioural responses of the Tribolium confusum to 6 blends of cereal volatiles. There were tested four doses (1 ng/min, 10 ng/min, 100 ng/min and 1000 ng/min in 50 μl of hexane applied on filter paper). A Y‐tube experiment revealed that T. confusum females and males were attracted to the blend 1, 4 and 5 at the concentrations between 1 and 10 ng/min. Confused T. confusum females and males were not attracted to any concentration of blend 2 and 3. Yet the beetle females and males were repelled by the highest concentrations (1000 ng/min) for all the blends tested and also by the concentration 100 ng/min of all the blends tested, except for blend 1 and 4. Females and males were also repelled by the three blend 6 concentrations tested (10, 100 and 1000 ng/min).     

Tribolium castaneum as a whole‐animal screening system for the detection and characterization of neuroprotective substances

Parkinson's disease (PD) is a movement disorder caused by the progressive loss of dopaminergic neurons. Natural antioxidants and plant extracts with neuroprotective properties offer a promising new therapeutic approach for PD patients, but a suitable large‐scale screening system is required for their discovery and preclinical analysis. Here we used the red flour beetle (Tribolium castaneum ) as a whole‐animal screening system for the detection and characterization of neuroprotective substances. Paraquat was added to the diet of adult beetles to induce PD‐like symptoms, which were quantified using a novel positive geotaxis behavioral assay. These paraquat‐induced behavioral changes were reduced in beetles fed on diets supplemented with l‐ dihydroxyphenylalanine, ascorbic acid, curcumin, hempseed flour, or the Chinese herb gou‐teng. T. castaneum is, therefore, a valuable model for the screening of neuroprotective substances in chemical libraries and plant extracts and could be developed as a model for the preclinical testing of therapeutic candidates for the treatment of neurodegenerative diseases, such as PD.     

Effects of caffeine on mating behavior and sperm precedence in Tribolium castaneum

Biogenic amines such as dopamine are physiologically neuroactive substances that affect behavioral and physiological traits in invertebrates, and it has long been known that these substances affect mating behavior in insects. Caffeine is a dopamine activator and thus enhances dopamine receptor activity. However, the effects of caffeine intake on insect mating behavior have been largely unexplored. Therefore, we examined the effect of caffeine on mating behavior in the red flour beetle Tribolium castaneum. Caffeine, which activates dopamine, affected the mating behavior of T. castaneum males. Males who orally ingested caffeine courted faster than males who did not, resulting in faster mounting of females and less time to a male's external aedeagus protrusion. However, the present results showed no difference in sperm precedence measured as a P2 value between males fed caffeine and males not fed caffeine. We discuss the effects of caffeine on insect mating and the possibility that caffeine consumption may cause males to mate with more females in the laboratory.     

Functional analysis of TcCTLP-5C2, a chymotrypsin-like serine protease needed for molting in Tribolium castaneum

In a previous study, we have characterized a gene family encoding chymotrypsin-like proteases from the red flour beetle, Tribolium castaneum (TcCTLPs). We identified 14 TcCTLP genes that were predominantly expressed in the midgut, where they presumably function in digestion. Two genes (TcCTLP-6C and TcCTLP-5C₂), however, additionally showed considerable expression in the carcass, and RNAi studies demonstrated that they are required for molting (Broehan et al., 2010; Insect Biochem. Mol. Biol 40, 274-83). Thus, the enzyme has distinct functions in different physiological environments. To study molecular adaptations that facilitate enzyme function in different environments, we performed an in-depth analysis of the molecular and enzymatic properties of TcCTLP-5C_2. We expressed different mutated versions of TcCTLP-5C₂ in form of factor Xa activatable pro-enzymes in insect cells using a baculoviral expression system, and purified the recombinant proteins by affinity chromatography. By measuring and comparing the enzyme activities, we obtained information about the significance of single amino acid residues in motifs that determine substrate specificity and pH tolerance. Further, we showed that TcCTLP-5C₂ is modified by N-glycosylation at amino acid position N137, which lies opposite to the catalytic cleft. Comparison of the enzymatic properties of non-glycosylated and glycosylated TcCTLP-5C₂ versions showed that N-glycosylation decreases V_max (maximum velocity) and k_cat (turnover) while leaving the K_m (specificity) unchanged. Thus, we provide evidence that N-glycosylation alters catalytic behavior by allosteric effects presumably due to altered structural dynamics as observed for chemically glycosylated enzymes.     

Mechanisms for tolerance to diatomaceous earth between strains of Tribolium castaneum

Fourteen strains of Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) had mortalities ranging from 5 to 100% when exposed to diatomaceous earth at 600 ppm for seven days. The most tolerant strain had a lethal dose for 50% of the population (LD₅₀) of 413 ppm and the most susceptible strain had a LD₅₀ of 238 ppm. Adults of the tolerant strain were lighter (2.0 mg) than the susceptible strain (2.6 mg). Tolerant adults lost water at lower rate (6 g h^–1 than susceptible adults (12 g h^–1), when held in wheat treated with 600 ppm diatomaceous earth for 24 h, than held at 5% r.h. with no food. Tolerant adults that were not exposed to diatomaceous earth lost water at a lower rate (3 g h^–1) than susceptible adults (5 g h^–1). Both strains, exposed and not exposed to diatomaceous earth died when their water content was between 33 and 37% of their total weight. Insects taken directly from the cultures had 52% (tolerant) and 53% (susceptible) of their total weight as water. Tolerant adults moved slower through grain and across filter paper than susceptible adults. Tolerant adults avoided wheat treated with diatomaceous earth at concentrations as low as 75 ppm, whereas the adults from the susceptible strain did not avoid diatomaceous earth, even at 600 ppm. The consequences of a strain tolerant to diatomaceous earth is discussed with respect to the use of diatomaceous earth to control stored-product insect infestations.     

Genetic variation and co-variation for fitness between intra-population and inter-population backgrounds in the red flour beetle, Tribolium castaneum

Hybrids from crosses between populations of the flour beetle, Tribolium castaneum, express varying degrees of inviability and morphological abnormalities. The proportion of allopatric population hybrids exhibiting these negative hybrid phenotypes varies widely, from 3% to 100%, depending upon the pair of populations crossed. We crossed three populations and measured two fitness components, fertility and adult offspring numbers from successful crosses, to determine how genes segregating within populations interact in inter-population hybrids to cause the negative phenotypes. With data from crosses of 40 sires from each of three populations to groups of five dams from their own and two divergent populations, we estimated the genetic variance and covariance for breeding value of fitness between the intra- and inter-population backgrounds and the sire × dam population interaction variance. The latter component of the variance in breeding values estimates the change in genic effects between backgrounds owing to epistasis. Interacting genes with a positive effect, prior to fixation, in the sympatric background but a negative effect in the hybrid background cause reproductive incompatibility in the Dobzhansky-Muller speciation model. Thus, the sire × dam population interaction provides a way to measure the progress towards speciation of genetically differentiating populations on a trait by trait basis using inter-population hybrids.     

Correlated effects of the response to conditioned medium in the flour beetle,Tribolium castaneum

Dispersal rates were measured in lines selected for high and low response of egg production to conditioned media, and responsivness of egg production was measured in lines selected for high and low dispersal. A positive correlation was found between these two traits, each of which had previously been found to have a simple genetic basis. It is suggested that in Tribolium castaneum the sensitivity to environmental conditions is mediated through a Sensor gene, which can activate either response, according to circumstances.     

The spatiotemporal dynamics of Tribolium castaneum (Herbst): adult flight and gene flow

Tribolium castaneum (Herbst) has been used as a model organism to develop and test important ecological and evolutionary concepts and is also a major pest of grain and grain products globally. This beetle species is assumed to be a good colonizer of grain storages through anthropogenic movement of grain, and active dispersal by flight is considered unlikely. Studies using T. castaneum have therefore used confined or walking insects. We combine an ecological study of dispersal with an analysis of gene flow using microsatellites to investigate the spatiotemporal dynamics and adult flight of T. castaneum in an ecological landscape in eastern Australia. Flying beetles were caught in traps at grain storages and in fields at least 1 km from the nearest stored grain at regular intervals for an entire year. Significantly more beetles were trapped at storages than in fields, and almost no beetles were caught in native vegetation reserves many kilometres from the nearest stored grain. Genetic differentiation between beetles caught at storages and in fields was low, indicating that although T. castaneum is predominantly aggregated around grain storages, active dispersal takes place to the extent that significant gene flow occurs between them, mitigating founder effects and genetic drift. By combining ecological and molecular techniques, we reveal much higher levels of active dispersal through adult flight in T. castaneum than previously thought. We conclude that the implications of adult flight to previous and future studies on this model organism warrant consideration.     

磷化氢对赤拟谷盗成虫体内CAT和SOD活性的影响

用赤拟谷盗Tribolium castaneum(Herbst)成虫,研究PH3对其体内超氧化物岐化酶(SOD)和过氧化氢酶(CAT)活性的影响。结果表明PH3熏蒸后,4种不同地理种群的成虫体内,CAT酶活性都有所降低。种群NGD1降低最多,接近57%,而NGD4则只降低了19%,NGD2和NGD3的降低都在40%左右。不同地理种群的成虫体内,SOD酶活性在熏蒸后都有所增加,种群NGD1增加幅度最大,NGD2增加幅度最小。SOD和CAT活性的变化幅度与成虫对PH3产生的抗性有关,抗性越大,酶活性变化越大。