Influences of elevated CO2 and pest damage on the allocation of plant defense compounds in Bt‐transgenic cotton and enzymatic activity of cotton aphid

Abstract Plant allocation to defensive compounds by elevated CO₂‐grown non‐transgenic and transgenic Bt cotton in response to infestation by cotton aphid, Aphis gossypii (Glover) in open‐top chambers under elevated CO₂ were studied. The results showed that significantly lower foliar nitrogen concentration and Bt toxin protein occurred in transgenic Bt cotton with and without cotton aphid infestation under elevated CO_2. However, significantly higher carbon/nitrogen ratio, condensed tannin and gossypol were observed in transgenic Bt cotton “GK‐12” and non‐transgenic Bt cotton ‘Simian‐3’ under elevated CO_2. The CO₂ level and cotton variety significantly influenced the foliar nitrogen, condensed tannin and gossypol concentrations in the plant leaves after feeding by A. gossypii. The interaction between CO₂ level × infestation time (24 h, 48 h and 72 h) showed a significant increase in cotton condensed tannin concentrations, while the interaction between CO₂ level × cotton variety significantly decreased the true choline esterase (TChE) concentration in the body of A. gossypi. This study exemplified the complexities of predicting how transgenic and non‐transgenic plants will allocate defensive compounds in response to herbivorous insects under differing climatic conditions. Plant defensive compound allocation patterns and aphid enzyme changes observed in this study appear to be broadly applicable across a range of plant and herbivorous insect interactions as CO₂ atmosphere rises.     

The lethal plant defense paradox remains: inducible host-plant aristolochic acids and the growth and defense of the pipevine swallowtail

Toxic plants with sequestering specialists are presented with a problem because plant derived toxins protect herbivores against natural enemies. It has been suggested that early induction of toxins and later relaxation of these defenses may help the plant resolve this problem because neonate caterpillars incur the physiological cost of dealing with toxins in early life, but are denied toxins when they are able to sequester them efficiently. In California, the pipevine swallowtail, Battus philenor L. (Lepidoptera: Papilionidae), feed exclusively on Aristolochia californica Torrey (Aristolochiaceae), an endemic vine that contains toxic alkaloids called aristolochic acids that caterpillars sequester to provide chemical defense in immature and adult stages. In a field experiment, the concentration of aristolochic acids doubled in the plant following leaf damage and returned to constitutive levels after six days. Neonate pipevine swallowtail caterpillars showed no aversion to high levels of aristolochic acid in a preference test. Caterpillars reared on leaves with supplemented aristolochic acid showed no physiological cost or increased mortality compared to caterpillars reared on un-supplemented leaves. Searching efficiency and capture rate of lacewing larvae (Chrysoperla), a common predator of first instar caterpillars, was compromised significantly after feeding on caterpillars reared on leaves with supplemented concentrations of aristolochic acid compared to caterpillars feeding on control plants. Additionally, mortality of lacewings increased when they were provided with a diet of B. philenor caterpillars reared on supplemented leaves compared to caterpillars reared on control leaves. Thus, the induction of aristolochic acids in the plant following leaf damage does not resolve the problem confronted by the plant and may confer benefits to this sequestering specialist.     

Brood defense and brood size in the great tit (Parus major): a test of a model of unshared parental investment

To understand the interaction of the many contextual variablesthat affect parental behavior a number of static optimalitymodels have been developed. Among these the one by Lazarus andInglis (1986) is the only one to specifically predict the magnitudeof unshared parental investment (PI), i.e., of parental carethat carries a cost to the parent and that benefits all currentoffspring equally because it cannot be divided among them. Weinvestigated specifically how parent great tits (Parus major)gear their brood defense, a form of unshared PI, to the sizeof the brood at stake and to the risk incurred as a functionof the type of predator. The predators used were dummies ofthe great spotted woodpecker (Picoides major) and of the tawnyowl (Strix aluco). Normally, adults can approach the woodpeckerwith impunity; it had inflicted heavy losses to nestlings ofthe study populations of great tits near Wolfsburg, Lower Saxony.Parent great tits whose brood had been artificially reducedto two young responded to the dummy with less defense than dida control group with their pre-test brood size left intact.The nature of defense was qualitatively the same as that elicitedby a live woodpecker. Parents confronting the owl near theirbrood decreased their response with an artificial reductionin brood size much less. Because the owl used poses a seriousrisk to the defenders, as compared to the woodpecker, the resultlends powerful support to the "total loss" version of the modelof unshared PI; it predicts brood size to affect unshared PImore strongly when there is less risk to the parent. This interpretationis correct to the extent that one premise of the model, namelythat of uncompromised parentage, can be relaxed; great tit broodscontain a sizeable number of extrapair young. Males defendedtheir brood more strongly than did females. Sex and brood manipulationadded up linearly when affecting defense level, i.e., therewas no interaction.     

Evolution of latex and its constituent defensive chemistry in milkweeds (Asclepias): a phylogenetic test of plant defense escalation

A tremendous diversity of plants exude sticky and toxic latex upon tissue damage, and its production has been widely studied as a defensive adaptation against insect herbivores. Here, we address variation in latex production and its constituent chemical properties (cardenolides and cysteine proteases) in 53 milkweeds [Asclepias spp. (Apocynaceae)], employing a phylogenetic approach to test macroevolutionary hypotheses of defense evolution. Species were highly variable for all three traits, and they showed little evidence for strong phylogenetic conservatism. Latex production and the constituent chemical defenses are thus evolutionarily labile and may evolve rapidly. Nonetheless, in phylogenetically independent analyses, we show that the three traits show some correlations (and thus share a correlated evolutionary history), including a positive correlation between latex exudation and cysteine protease activity. Conversely, latex exudation and cysteine protease activity both showed a trade‐off with cardenolide concentrations in latex. We also tested whether these traits have increased in their phenotypic values as the milkweeds diversified, as predicted by plant defense escalation theory. Alternative methods of testing this prediction gave conflicting results – there was an overall negative correlation between amount of evolutionary change and amount of latex exudation; however, ancestral state reconstructions indicated that most speciation events were associated with increases in latex. We conclude by (i) summarizing the evidence of milkweed latex itself as a multivariate defense including the amount exuded and toxin concentrations within, (ii) assessing the coordinated evolution of latex traits and how this fits with our previous notion of ‘plant defense syndromes’, and finally, (iii) proposing a novel hypothesis that includes an ‘evolving community of herbivores’ that may promote the escalation or decline of particular defensive strategies as plant lineages diversify.     

The spitting image of plant defenses: Effects of plant secondary chemistry on the efficacy of caterpillar regurgitant as an anti‐predator defense

In the arms race between plants, herbivores, and their natural enemies, specialized herbivores may use plant defenses for their own benefit, and variation in plant traits may affect the benefits that herbivores derive from these defenses. Pieris brassicae is a specialist herbivore of plants containing glucosinolates, a specific class of defensive secondary metabolites. Caterpillars of P. brassicae are known to actively spit on attacking natural enemies, including their main parasitoid, the braconid wasp Cotesia glomerata. Here, we tested the hypothesis that variation in the secondary metabolites of host plants affects the efficacy of caterpillar regurgitant as an anti‐predator defense. Using a total of 10 host plants with different glucosinolate profiles, we first studied natural regurgitation events of caterpillars on parasitoids. We then studied manual applications of water or regurgitant on parasitoids during parasitization events. Results from natural regurgitation events revealed that parasitoids spent more time grooming after attack when foraging on radish and nasturtium than on Brassica spp., and when the regurgitant came in contact with the wings rather than any other body part. Results from manual applications of regurgitant showed that all parameters of parasitoid behavior (initial attack duration, attack interruption, grooming time, and likelihood of a second attack) were more affected when regurgitant was applied rather than water. The proportion of parasitoids re‐attacking a caterpillar within 15 min was the lowest when regurgitant originated from radish‐fed caterpillars. However, we found no correlation between glucosinolate content and regurgitant effects, and parasitoid behavior was equally affected when regurgitant originated from a glucosinolate‐deficient Arabidopsis thaliana mutant line. In conclusion, host plant affects to a certain extent the efficacy of spit from P. brassicae caterpillars as a defense against parasitoids, but this is not due to glucosinolate content. The nature of the defensive compounds present in the spit remains to be determined, and the ecological relevance of this anti‐predator defense needs to be further evaluated in the field.     

In situ modification of herbivore-induced plant odors: a novel approach to study the attractiveness of volatile organic compounds to parasitic wasps

Many parasitic wasps (parasitoids) exploit volatile organic compounds (VOCs) emitted by herbivore-infested plants in order to locate their hosts, but it remains largely unknown which specific compounds within the volatile blends elicit the attractiveness to parasitoids. One way of studying the importance of specific VOCs is to test the attractiveness of odor blends from which certain compounds have been emitted. We used this approach by testing the attraction of naive and experienced females of the two parasitoids Cotesia marginiventris and Microplitis rufiventris to partially altered volatile blends of maize seedlings (Zea mays var. Delprim) infested with Spodoptera littoralis larvae. Adsorbing filter tubes containing carbotrap-C or silica were installed in a four-arm olfactometer between the odor source vessels and the arms of the olfactometer. The blends breaking through were tested for chemical composition and attractiveness to the wasps. Carbotrap-C adsorbed most of the sesquiterpenes, but the breakthrough blend remained attractive to naive C. marginiventris females. Silica adsorbed only some of the more polar VOCs, but this essentially eliminated all attractiveness to naive C. marginiventris, implying that among the adsorbed compounds there are some that play key roles in the attraction. Unlike C. marginiventris, M. rufiventris was still attracted to the latter blend, showing that parasitoids with a comparable biology may employ different strategies in their use of plant-provided cues to locate hosts. Results from similar experiments with modified odor blends of caterpillar-infested cowpea (Vigna unguiculata) indicate that key VOCs in different plant species vary greatly in quality and/or quantity. Finally, experienced wasps were more strongly attracted to a specific blend after they perceived the blend while ovipositing in a host. Considering the high number of distinct adsorbing materials available today, this in situ modification of complex volatile blends provides a new and promising approach pinpointing on key attractants within these blends. Advantages and disadvantages compared to other approaches are discussed.     

Sick plants in grassland communities: a growth‐defense trade‐off is the main driver of fungal pathogen abundance

Aboveground fungal pathogens can substantially reduce biomass production in grasslands. However, we lack a mechanistic understanding of the drivers of fungal pathogen infection and impact. Using a grassland global change and biodiversity experiment we show that the trade‐off between plant growth and defense is the main determinant of infection incidence. In contrast, nitrogen addition only indirectly increased incidence via shifting plant communities towards faster growing species. Plant diversity did not decrease incidence, likely because spillover of generalist pathogens or dominance of susceptible plants counteracted negative diversity effects. A fungicide treatment increased plant biomass production and high levels of infection incidence were associated with reduced biomass. However, pathogen impact was context dependent and infection incidence reduced biomass more strongly in diverse communities. Our results show that a growth‐defense trade‐off is the key driver of pathogen incidence, but pathogen impact is determined by several mechanisms and may depend on pathogen community composition.     

Ecological role of reindeer summer browsing in the mountain birch (<Emphasis Type="Italic">Betula pubescens</Emphasis> ssp. <Emphasis Type="Italic">czerepanovii</Emphasis>) forests: effects on plant defense,litter decomposition,and soil nutrient cycling

Mammalian herbivores commonly alter the concentrations of secondary compounds in plants and, by this mechanism, have indirect effects on litter decomposition and soil carbon and nutrient cycling. In northernmost Fennoscandia, the subarctic mountain birch (Betula pubescens ssp. czerepanovii) forests are important pasture for the semidomestic reindeer (Rangifer tarandus). In the summer ranges, mountain birches are intensively browsed, whereas in the winter ranges, reindeer feed on ground lichens, and the mountain birches remain intact. We analyzed the effect of summer browsing on the concentrations of secondary substances, litter decomposition, and soil nutrient pools in areas that had been separated as summer or winter ranges for at least 20 years, and we predicted that summer browsing may reduce levels of secondary compounds in the mountain birch and, by this mechanism, have an indirect effect on the decomposition of mountain birch leaf litter and soil nutrient cycling. The effect of browsing on the concentration of secondary substances in the mountain birch leaves varied between different years and management districts, but in some cases, the concentration of condensed tannins was lower in the summer than in the winter ranges. In a reciprocal litter decomposition trial, both litter origin and emplacement significantly affected the litter decomposition rate. Decomposition rates were faster for the litter originating from and placed into the summer range. Soil inorganic nitrogen (N) concentrations were higher in the summer than in the winter ranges, which indicates that reindeer summer browsing may enhance the soil nutrient cycling. There was a tight inverse relationship between soil N and foliar tannin concentrations in the winter range but not in the summer range. This suggests that in these strongly nutrient-limited ecosystems, soil N availability regulates the patterns of resource allocation to condensed tannins in the absence but not in the presence of browsing.     

Selective consumption of acorns by the Japanese wood mouse according to tannin content: a behavioral countermeasure against plant secondary metabolites

We examined the presence of selective consumption against tannins in acorns as a pre-ingestive countermeasure to plant secondary metabolites by using the Japanese wood mouse (Apodemus speciosus) and acorns of Quercus serrata, which contained ca. 6.4% tannins on a dry weight basis. In addition, the presence of selective consumption against proteins was also examined. In the acorn-feeding experiment, 18 wood mice were allocated to two groups: the experienced group (N = 9), which had previous experience in feeding on acorns, and the inexperienced group (N = 9), which had no experience. Mice of both groups were fed only acorns for 3 nights. Selectivity against tannins (an index estimated from the tannin content in control, remaining, and ingested acorns) was significantly positive in the experienced group, indicating the presence of selective consumption against tannins. In contrast, mice in the inexperienced group did not show significant selectivity against tannins. Comparing the selectivity indices directly between two groups, however, they did not differ significantly. Selective consumption against proteins rather than that for proteins also occurred in the experienced group, but it was thought to be a byproduct resulted from the selectivity against tannins. Selective consumption against tannins can mitigate the negative effects of tannins by decreasing tannin intake.     

Manipulation of the hypocotyl sink activity by reciprocal grafting of two Raphanus sativus varieties: its effects on morphological and physiological traits of source leaves and whole‐plant growth

To reveal whether hypocotyl sink activities are regulated by the aboveground parts, and whether physiology and morphology of source leaves are affected by the hypocotyl sink activities, we conducted grafting experiments using two Raphanus sativus varieties with different hypocotyl sink activities. Comet (C) and Leafy (L) varieties with high and low hypocotyl sink activities were reciprocally grafted and resultant plants were called by their scion and stock such as CC, LC, CL and LL. Growth, leaf mass per area (LMA), total non‐structural carbohydrates (TNCs) and photosynthetic characteristics were compared among them. Comet hypocotyls in CC and LC grew well regardless of the scions, whereas Leafy hypocotyls in CL and LL did not. Relative growth rate was highest in LL and lowest in CC. Photosynthetic capacity was correlated with Rubisco (ribulose 1·5‐bisphosphate carboxylase/oxygenase) content but unaffected by TNC. High C/N ratio and accumulation of TNC led to high LMA and structural LMA. These results showed that the hypocotyl sink activity was autonomously regulated by hypocotyl and that the down‐regulation of photosynthesis was not induced by TNC. We conclude that the change in the sink activity alters whole‐plant growth through the changes in both biomass allocation and leaf morphological characteristics in R. sativus.     

Multiple source pools for Galápagos plant species richness: a critical analysis of the line of sight connectivity index

An index (C_i*E) combining the number of line‐of‐sight islands (C_i) within a radius i and target island elevation (E) has been proposed as an improved predictive model of plant species richness (S_t) in the Galápagos Archipelago. We examined this index critically and found that several major flaws preclude it from being a useful predictive tool for the archipelago. Although the number of collecting trips to an island was reported over 20 years ago to have substantial predictive value for reported plant species richness in the Galápagos Islands, this relationship was ignored in multiple regression analyses of the index. When we included the number of collecting trips in different multiple regression analyses of the index, C_i*E had less predictive power than collecting trips or ceased to be significant at all. Additionally, the strong significant relationship between elevation and area in the Galápagos Archipelago results in area having a major confounding influence on the C_i*E index. When elevation is removed from the C_i*E index, the predictive power of C_i is far less than area alone. Finally, the data used to construct and correlate the C_i*E index with (S_t) were based only on a subset of the islands and species lists that were incomplete or out of date. Species richness on islands can be related to the interaction of different factors, so development and testing of indices like C_i*E is not inappropriate. However, it is important to examine the interrelationships among the components of these indices thoroughly, and not ignore the effect of factors already known to have high predictive power. We propose several ways in which more meaningful indices of source pool(s) capacity can be constructed.     

A convenient screening test system and a model for thermal germination responses of wild plant seeds: behaviour of model and real seeds in the system

Abstract A convenient test system for screening the thermal germination behaviour of seeds was developed for both basic and applied research in seed germination ecophysiology. Only two temperature-controlled facilities, a test period of about a month and a relatively small number of sample seeds arc needed to obtain information on the thermal-germination parameters of individual seed populations, such as lower or higher limit temperatures, and thermal times required for germination. In the test system, the germination performances of sample seed populations were compared under two temperature regimes: a gradually increasing temperature regime and a gradually decreasing temperature regime, in which the seeds were subjected to gradually changing temperatures in the range of 4 36°C. In order to assess the effects of various values for thermal-germination parameters on the patterns of germination performance in the system, the behaviour of model seeds characterized by a definite set of thermal germination parameters were investigated. Referring to the results of the simulations, the actual germination patterns of some wild-seed populations in the test system were interpreted in terms of thermal-germination parameters.     

Orange roughy otolith growth rates: a direct experimental test of the Romanek–Gauldie otolith growth model

Endolymph chemistry (Ca²⁺, pH, Na⁺) was measured in 17 orange roughy (Hoplostethus atlanticus; Teleostei; Trachichthyidae). Theoretical daily microincrement widths based on the Romanek and Gauldie model of otolith growth were calculated from the measure of otolith chemistry. Theoretical daily microincrement widths were well-correlated (r²=59%) with the width of the last daily microincrement growing at the posterior edge of otoliths taken from the sampled fish.     

Carbon dynamics of terrestrial ecosystems on the Tibetan Plateau during the 20th century: an analysis with a process‐based biogeochemical model

Aim The Tibetan Plateau accounts for about a quarter of the total land area of China and has a variety of ecosystems ranging from alpine tundra to evergreen tropics. Its soils are dominated by permafrost and are rich in organic carbon. Its climate is unique due to the influence of the Asian monsoon and its complex topography. To date, the carbon dynamics of the Tibetan Plateau have not been well quantified under changes of climate and permafrost conditions. Here we use a process‐based biogeochemistry model, the Terrestrial Ecosystem Model (TEM), which was incorporated with a soil thermal model, to examine the permafrost dynamics and their effects on carbon dynamics on the plateau during the past century. Location The Tibetan Plateau. Methods We parameterize and verify the TEM using the existing data for soil temperature, permafrost distribution and carbon and nitrogen from the region. We then extrapolate the model and parameters to the whole plateau. Results During the 20th century, the Tibetan Plateau changed from a small carbon source or neutral in the early part of the century to a sink later, with a large inter‐annual and spatial variability due to changes of climate and permafrost conditions. Net primary production and soil respiration increased by 0.52 and 0.22 Tg C year^?1, respectively, resulting in a regional carbon sink increase of 0.3 Tg C year^?1. By the end of the century, the regional carbon sink reached 36 Tg C year^?1 and carbon storage in vegetation and soils is 32 and 16 Pg C, respectively. On the plateau, from west to east, the net primary production, soil respiration and net ecosystem production increased, due primarily to the increase of air temperature and precipitation and lowering elevation. In contrast, the decrease of carbon fluxes from south to north was primarily controlled by precipitation gradient. Dynamics of air temperature and associated soil temperature and active layer depth resulted in a higher plant carbon uptake than soil carbon release, strengthening the regional carbon sink during the century. Main conclusions We found that increasing soil temperature and deepening active layer depth enhanced soil respiration, increasing the net nitrogen mineralization rate. Together with the effects of warming air temperature and rising CO₂ concentrations on photosynthesis, the stronger plant nitrogen uptake due to the enhanced available nitrogen stimulates plant carbon uptake, thereby strengthening the regional carbon sink as the rate of increase net primary production was faster than that of soil respiration. Further, the warming and associated soil thermal dynamics shifted the regional carbon sink from the middle of July in the early 20th century to early July by the end of the century. Our study suggests that soil thermal dynamics should be considered for future quantification of carbon dynamics in this climate‐sensitive region.     

Geographic and stratigraphic change in the morphology of Triarthrus beckii (Green) (Trilobita): a test of the Plus ça change model of evolution

The Plus ça change model predicts that deepwater trilobite species such as Triarthrus should exhibit gradual phyletic evolution. A detailed stratigraphic sequence of Triarthrus beckii specimens considered together with geographically separated samples from a single time interval provide a test of the Plus ça change model. We examined geographic patterns of variation in cranidial shape based on specimens from four approximately synchronous levels within the Upper Ordovician (lower Edenian) strata of New York, Québec, Kentucky and Pennsylvania. All geographic populations differ in mean size, except for New York and Pennsylvania. Because allometry is present in both meraspid and holaspid phases of the trilobite, size effects on shape were removed by ontogenetic standardization. Cranidial shapes were then compared among the four study sites and eight stratophenetic samples from New York, obtained by subdividing our Mohawkian Composite Standard Section into 20-m-thick intervals. Goodall's F-test of pairwise comparisons of cranidial shape between eight subintervals in New York and the other three populations are all significant. Geographic variation in cranidial size and shape in T . beckii appears to display a gradient or cline-like pattern that is related to paleogeography. Although cranidial shape is not entirely static within the New York stratophenetic series, it is in all cases different from that sampled elsewhere in the region. Thus, there is no evidence of wholesale immigration and emigration among the geographic areas. Instead, the geographic gradient of T . beckii shape variation may have remained fairly stable over ca. 3 million years during the mid-Chatfieldian to early Edenian interval. This result is indicative of stasis. Consequently, the evolutionary history of T . beckii contradicts the Plus ça change model.     

Use of a generalised linear model to test habitat preferences: the example of Zingel asper, an endemic endangered percid of the River Rhône

SUMMARY 1. A study of microhabitat preferences was conducted on Zingel asper , an endangered endemic species from the Rhône catchment. A generalised linear model allowed us to test statistically the non-random habitat selection and the effect of season and site on this habitat selection.
2. The analysis detected significant preferences for the three physical variables considered: water depth, water velocity and substratum size.
3. A seasonal shift in the substratum size preference was found: preference for stones increasing during the spawning season. Depth preference varied between sites, suggesting a possible plasticity in habitat selection.
4. These results suggest that the availability of suitable physical habitat plays a significant role in determining fish distribution in the River Beaume.     

Host plant preference and performance of the sibling species of butterflies <Emphasis Type="Italic">Leptidea sinapis</Emphasis> and <Emphasis Type="Italic">Leptidea reali</Emphasis>: a test of the trade-off hypothesis for food specialisation

A large proportion of phytophagous insect species are specialised on one or a few host plants, and female host plant preference is predicted to be tightly linked to high larval survival and performance on the preferred plant(s). Specialisation is likely favoured by selection under stable circumstances, since different host plant species are likely to differ in suitability—a pattern usually explained by the “trade-off hypothesis”, which posits that increased performance on a given plant comes at a cost of decreased performance on other plants. Host plant specialisation is also ascribed an important role in host shift speciation, where different incipient species specialise on different host plants. Hence, it is important to determine the role of host plants when studying species divergence and niche partitioning between closely related species, such as the butterfly species pair Leptidea sinapis and Leptidea reali. In Sweden, Leptidea sinapis is a habitat generalist, appearing in both forests and meadows, whereas Leptidea reali is specialised on meadows. Here, we study the female preference and larval survival and performance in terms of growth rate, pupal weight and development time on the seven most-utilised host plants. Both species showed similar host plant rank orders, and larvae survived and performed equally well on most plants with the exceptions of two rarely utilised forest plants. We therefore conclude that differences in preference or performance on plants from the two habitats do not drive, or maintain, niche separation, and we argue that the results of this study do not support the trade-off hypothesis for host plant specialisation, since the host plant generalist Leptidea sinapis survived and performed as well on the most preferred meadow host plant Lathyrus pratensis as did Leptidea reali although the generalist species also includes other plants in its host range. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A validated HPLC method for the determination of dimethyl‐4,4′‐dimethoxy‐5,6,5′,6′‐dimethylene dioxybiphenyl‐2,2′‐dicarboxylate (DDB) with fluorescence detection in raw material and pill form: application to an in vitro dissolution test and a content uniformity test

A simple, sensitive and rapid HPLC method with fluorescence detection for the determination of dimethyl‐4,4′‐dimethoxy‐5,6,5′,6′‐dimethylene dioxybiphenyl‐2,2′‐dicarboxylate (DDB) in the raw material and pill form was developed. Liquid chromatography was performed on a C₁₈ column (250 × 4.6 mm i.d., 5 µm particle size), the mobile phase consisted of methanol and 0.05 M sodium dihydrogen phosphate buffer (80 : 20, v/v), and the apparent pH of the mobile phase was adjusted to 3. The fluorescence detector was operated at excitation/emission wavelengths of 275/400 nm. The proposed method allows the determination of DDB within concentration range 0.1–1.5 µg/mL with a limit of detection of 0.032 µg/mL, a limit of quantification of 0.097 µg/mL and a correlation coefficient of 0.9997. The proposed method has been successfully applied for the analysis of DDB in its pills with a percentage recovery of 98.45 ± 0.32. The method was fully validated according to ICH guidelines. Moreover, the high sensitivity of the method permits its use in an in vitro dissolution test for DDB under simulated intestinal conditions. In addition, the proposed method was extended to a content uniformity test according to USP guidelines. Copyright © 2014 John Wiley & Sons, Ltd.