Ontogenic Stages Research Articles

Characterization of Kiss/Kissr system and expression profiling through developmental stages indicate kiss1 to be the active isotype in Clarias magur.

Kisspeptin (Kiss) and kisspeptin receptor (Kissr) system is a key regulator of GnRH expression in several vertebrates. The Indian catfish, Clarias magur, is popular in the Indian sub-continent, and a neo-type of the Asian catfish, C. batrachus. Catfish breeding is constrained as males do not release milt captivity with/without stimulation. Magur Kiss/Kissr system comprising of kiss1, kiss2, kissr1, and kissr2 genes was characterized for the first time. Full-length mRNA was sequenced using RACE PCR. Neighbor-joining tree of predicted proteins shows one clade of teleost orthologs. Magur whole genome (NCBI GenBank) has single copies of each gene, though yet unannotated/misannotated. Anomalies in the nomenclature of earlier sequences in GenBank were noted. Relative gene expression was profiled during various ontogenic stages, in six tissues including brain and gonads at maturity, and also in brains and gonads of premature and spent fish. Expression of gnrh1, gnrhr1, and gnrhr2 was estimated concomitantly. The kiss1 was the first to be twofold upregulated (P < 0.05) at 12h post fertilization. Kiss/Kissr genes expressed primarily in the brain, ovary, and testis. Though kiss2 was 10 times higher than kiss1, only kiss1 showed significant modulation across stages and appears to be the active isotype that regulates GnRH in magur.

Putting space into trait ecology: Trait, environment and biodiversity relationships at multiple spatial scales

Abstract Ecological processes such as environmental filtering and biotic interactions that shape species' traits and community diversity often vary with geographic distance, potentially generating spatial structures in trait variation, covariation and biodiversity data. Understanding spatial structures of trait, environment and biodiversity, or the spatial link between those factors, is fundamental to identifying spatially explicit assembly processes or biodiversity distributions in spatially heterogeneous landscapes but remains unclear. To address the issue, we gathered individual‐level leaf and diameter traits data paired with environmental data from a 4.8 ha subtropical Chinese forest and divided the forest into 25, 100, 400 and 1936 m2 grids representing contrasting spatial grains. Using Moran's correlograms, we quantified the spatial structures of trait variation and covariation, environmental conditions and biodiversity. We assessed the links between those variables using path analyses. Most variables were spatially positively autocorrelated. However, trait mean was more autocorrelated than trait variation or covariation, and intraspecific trait variation was more autocorrelated than interspecific variation. Autocorrelations in those community properties were generally weak at the large grain. Path analyses indicated positive associations between interspecific trait variation and species diversity at a very small to medium scale and a positive association between intraspecific variation and small‐scale functional diversity. Trait covariation constrained biodiversity, and multi‐trait means were negatively linked to very small‐ to medium‐scale species diversity but positively to medium‐ and large‐scale functional diversity. Patterns regarding multi‐trait community structure–environment–biodiversity associations were generally held for individual traits. However, depending on the trait, spatial scale and plant ontogenic stage, the pattern's strength changed, or occasionally, their sign reversed. We attribute spatial patterns in multi‐trait mean and covariation to scale‐dependent variation in environmental heterogeneity and trait variation to scale‐dependent competition. Synthesis. Our study provides novel insights into spatial and scale‐dependent variability in functional community structure, environment and biodiversity, and their relationships. Our results demonstrate the usefulness of spatial trait analyses in identifying scale‐dependent assembly processes or finding the importance of processes to biodiversity distributions in spatially heterogeneous landscapes. A spatially explicit perspective is thus helpful for the progress of trait ecology.

Ontogenic stage-associated SA response contributes to leaf age-dependent resistance in Arabidopsis and cotton.

As leaves grow, they transition from a low-microbe environment embedded in shoot apex to a more complex one exposed to phyllosphere microbiomes. Such change requires a coordinated reprogramming of cellular responses to biotic stresses. It remains unclear how plants shift from fast growth to robust resistance during organ development. Here, we reported that salicylic acid (SA) accumulation and response were temporarily increased during leaf maturation in herbaceous annual Arabidopsis. Leaf primordia undergoing active cell division were insensitive to the elicitor-induced SA response. This age-dependent increase in SA response was not due to prolonged exposure to environmental microbes. Autoimmune mutants with elevated SA levels did not alter the temporal pattern dependent on ontogenic stage. Young Arabidopsis leaves were more susceptible than mature leaves to Pseudomonas syringae pv. tomato (Pto) DC3000 cor- infection. Finally, we showed a broadly similar pattern in cotton, a woody perennial, where young leaves with reduced SA signaling were preferentially invaded by a Xanthomonas pathogen after leaf surface infection. Through this work, we provided insights in the SA-mediated ontogenic resistance in Arabidopsis and tomato.

Allelopathic effects of Eucalyptus extract and wood vinegar on germination and sprouting of rapeseed (Brassica rapa L.).

Eucalyptus plantations are usually characterized by low biodiversity due to allelopathy effects. Wood vinegar is considered a complex growth regulator that can promote plant growth at low concentrations. However, there is information scarcity about the co-application of eucalypt leaf water extract and wood vinegar on plants. This study aimed at clarifying whether wood vinegar can protect seed germination against suppression by eucalypt-induced allelopathy. We examined germination behavior and seedling elongation characteristics in rapeseed (Brassica rapa L.) treated with different solutions of wood vinegar and eucalypt leaf water extract. The results showed that eucalypt leaf water extracts, wood vinegar solutions, and their mixture allelopathically suppressed seed germination rate. After rapeseed sprouting, eucalypt leaf water extracts promoted root elongation, stem elongation, and fresh weight elongation. Malondialdehyde content was also lower under the influence of eucalypt leaf water extract. Mixture of high concentration of eucalypt leaf water extract and lower concentration of wood vinegar significantly promoted root elongation. Therefore, both eucalypt leaf water extract and wood vinegar are complex plant growth regulators, which can be used to inhibit or stimulate plants at different ontogenic stages. During the seed germination period, both eucalypt leaf extracts and wood vinegar could be used as weed inhibitors. Conversely, during the period of sprouting (seedling establishment), low concentrations of eucalypt leaf extracts and wood vinegar can promote growth.

MLL-AF9-Induced Leukaemia Changes during Ontogenic Stages

MLL-AF9 causes acute lymphoid leukaemia (ALL), acute myeloid leukaemia (AML) and mixed phenotype acute leukaemia (MPAL) in infants and children. Infant leukaemia originates in utero, yet this has never been formerly shown for MLL-AF9-driven disease. Multiple mouse models invariably revealed that MLL-AF9 can induce myelo-monocytic AML, however, they failed to recapitulate the ALL and MPAL phenotypes as presented in paediatric patients. To explore the impact of developmental stage for MLL-AF9 leukaemic burden and phenotypes, we established a new inducible (“Tet-off”), reversible, transplantable mouse model in which expression of the fusion is controlled by the 3' stem cell leukaemia (SCL)-enhancer element. We crossed SCL-tTA mice(1) with inducible MLL-AF9 mice(2) to generate double transgenic SCLtTA/iMLL-AF9 mice. To determine the impact of developmental stage, MLL-AF9 was induced from the conceptional stage for prenatal (E0), in neonates for postnatal (P0) and at 20 weeks for adult (≥20 weeks) cohorts. Following induction for 6-8 weeks in vivo MLL-AF9 expression was measured in haematopoietic stem cells (HSCs, (Lin-cKit+Sca-1+CD150+CD48-), lymphoid-myeloid primed progenitors (HPC-1/LMPPs, LSK CD150-CD48+/Flt3+), and granulocyte-monocyte progenitors (GMPs, Lin-cKit+Sca-1CD34+CD16/32hi). We found that MLL-AF9 expression levels were comparable within each population across the age groups but observed higher MLL-AF9 expression in the GMPs compared to HSCs in the postnatal model, concomitant with a moderate expansion in the GMP clonal size. Upon postnatal MLL-AF9 induction, all the mice developed leukaemia with a short latency of 13 weeks and complete penetrance. In contrast, E0 prenatal- or adult-induction resulted in longer latencies of 32 and 24 weeks respectively. Despite sustained MLL-AF9 expression, not all the mice developed leukaemia in prenatal- and adult-induced cohorts. Postnatal induced mice developed myelo-monocytic (AML-M4), megakaryoblastic (AML-M7), or MPAL, whereas prenatal and adult induction of MLL-AF9 developed AML-M4 exclusively. Regardless of leukaemia phenotypes, all diseased mice displayed thrombocytopenia and splenomegaly, and leucocytosis. Independent from the age at which MLL-AF9 was induced, all AML-M4 mice had clonal expansion of GMPs. Adult-induced AML-M4 mice had an increase in HSCs while postnatal-induced mice had an increase in HPC-1/LMPPs. In postnatal-induced MPAL mice, there was expansion of GMPs and HPC-1/LMPP populations, and postnatal-induced AML-M7 mice had clonal expansions of HSC, HPC-1/LMPP, and MEP populations with no expansion in GMPs. Thus, the age when the MLL-AF9 fusion is induced does not only determine the disease latency and penetrance, but also affects lineage determination, and the amount of leukaemic HSPCs. Leukaemic-GMP (L-GMP) and leukaemic-HSC (L-HSC) have been characterized in adult models as leukaemic stem cells (LSC). Transplantation of bulk and L-GMPs from adult-induced AML-M4 cells were able to cause AML with complete penetrance, in line with published data and validating our model. However, prenatal- and postnatal-induced AML-M4 cells were not transplantable. In all age cohorts, bulk AML cells formed serially plateable large and compact colonies and were reversible. Collectively, LSCs exist within a GMP-like population of adult-induced MLL-AF9 AML, they may however reside in different HSPC populations in prenatal- and postnatal-induced disease. In summary, this novel inducible model recapitulates several features of infant, paediatric and adult MLL-AF9 AML demonstrating that ontogenic stage is a determining factor in differences in disease biology.

Selective pressure on ontogenic stages of blue mussels (Mytilus edulis, L.)

Our previous work confirmed that the higher survival, growth, and commercial performance of a mussel stock collected from a specific lagoon have been maintained over the last 25 years at a microgeographic scale despite intensive transfers of its spat to other local mussel sites. The objective of this study was to identify potential selection mechanisms supporting the quality of this stock. We used a mesocosm approach to examine the effect of maximum seawater temperature measured in the field on the different ontogenic stages of Mytilus edulis to simulate similar patterns in the laboratory. We demonstrated that thermal selection did not affect the development of peri-metamorphic stages (< 1 mm) since no effects were observed on growth, survival, or prodissoconch II size under natural thermal variations. However, our experimental results clearly indicated the existence of a thermal selective pressure (24 °C for 85 h) on 4.5–7 mm juvenile mussels, with growth performance enhanced in surviving individuals. Mortality was 37% higher compared to the control group, suggesting the elimination of more sensitive and less performant mussels. Interestingly, surviving individuals showed improved shell and tissue growth rates, by 22 and 51%, respectively. Our results support the hypothesis that the thermal selection of highly performant stock is size-dependent. The selective pressure demonstrated in this study along with our previous work highlighting the physiological and genetic differences between local stocks constitute the key mechanisms leading to the observed stable production performance over decades at a microgeographic scale.

Competition and site weakly explain tree growth variability in undisturbed Central African moist forests

Abstract Identifying and quantifying factors that influence tree growth are crucial issues to ensure sustainable forest management, particularly in moist tropical forests. Tree growth depends on several factors comprising ontogenic stage, competition by neighbours and environmental conditions. Several studies have focused on one or two of them, but very few have considered all three, especially in Central Africa. We investigated the effects of diameter and competition on tree growth, in four Central African sites characterized by their soil physicochemical properties, at both tree community and population levels. We calibrated growth models using diameter data collected on 29,741 trees between 2015 and 2018, on twelve 4 or 9‐ha plots spread over the four sites. These models included diameter, wood density, competition indices and site effect as explainable variables at the community level and excluded wood density at the population level. At the community level, the best models explained 11% of growth variability with a decreasing effect of species wood density, diameter, site and competition. Our results show that even if low, site effect can result from different soil nutrients depending on both tree size and species wood density. We observed higher tree growth on sites with (i) high exchangeable K, organic C, total N and total P for low wood density species; (ii) high available P and C:N for small trees, high exchangeable Ca and Mg for medium to large trees, all belonging to medium and hard wood density species. At the population level, the best models explained between 0 to 43% of growth variability, with significant competition effect (resp. site effect) for 21 (resp. 9) of the 43 species studied. Site ranking varied greatly between the 9 species concerned, probably reflecting different sensitivities to the scarcity of particular soil nutrients. Synthesis. Our study provides original results on the factors influencing tree growth in Central Africa, showing that the potential effect of soil nutrients depends on tree size and species wood density. Remaining highly unpredictable at the population level, this effect makes it essential to increase the number of dynamics monitoring systems in logging concessions.

Architectural traits underlie growth form diversity and polycarpic versus monocarpic life histories in Cerberiopsis (Apocynaceae)

Abstract Plant architecture strongly influences plant growth habits, as it determines the arrangement, function and fate of meristems. How architecture could be involved in the monocarpic life history, i.e. dying after flowering, remains poorly investigated. Monocarpy is evident in some species since they are annual or because their single stem flowers apically. However, monocarpy in long-lived branched trees is rare and remains poorly understood. We aim to highlight the architectural features involved in the monocarpic strategy of Cerberiopsis candelabra, a rainforest tree endemic to New Caledonia. We conducted a comparative analysis of the genus, which comprises three species with different growth habits. Twenty plants of each species were studied at different ontogenic stages. We compared their developmental sequence and analysed their processes of growth, branching, flowering and reiteration. We identified a combination of traits that distinguish the species, and we found a syndrome of two architectural features that support the monocarpic strategy in C. candelabra: the synchronous flowering of all terminal meristems and the absence of delayed branching. Flowering in C. candelabra preferentially occurs when the complete architectural sequence is developed, but the plant never shows signs of senescence, suggesting that environmental stresses, such as wind disturbance, could be the main trigger for flowering. The architecture of C. candelabra is suggested to be the most derived in the genus.

Paleohistology of Caraguatypotherium munozi (Mammalia, Notoungulata, Mesotheriidae) from the early late Miocene of northern Chile: A preliminary ontogenetic approach.

The Miocene Caragua fossil fauna in northern Chile contains a considerable number (7) of articulated partial skeletons tentatively assigned to Caraguatypotherium munozi (Notoungulata, Mesotheriidae), which presents up to 40% body size difference. Since either inter- and intra- specific wide size range has been observed in the Mesotheriidae family in general, we wanted explore the ontogenic stage signature of the sample, by carrying out the first comprehensive paleohistological description of the appendicular system in Notoungulata. Results show that: 1) they can be classified as subadults and adults, based on the presence of bone tissues typical of ceased somatic growth; 2) there is a notorious inter-skeletal variation on bone growth rates (skeletal modularity), particularly, the humerus showed a slower diameter growth and less remodelling than the femur, resulting as a better element for ontogenetic analyses; 3) marked cyclical growth is observed, characterised by fast early ontogenic continuous growth, and subsequent fast/slow stratified bone tissue layering. In general, such growth pattern suggests that C. munozi had a similar ontogenetic growth process as other modern mammals, that it should also be influenced by other sex-related, ecological and environmental factors. Likely related to the presence of rapid climatic variations, due to orogenic uplift and concomitant re-organization of the drainage processes along the western tectonic front of the Central Andes at that time.

Do trait responses to simulated browsing in Quercus robur saplings affect their attractiveness to Capreolus capreolus the following year?

With the rise of large herbivore populations in most northern hemisphere forests, browsing is becoming an increasingly important driver of forest regeneration dynamics. Among other processes affecting the regeneration, the concept of plant-herbivore feedback loops holds that browsed saplings are more subject to subsequent herbivory. This phenomenon is interpreted as a consequence of compensatory growth following browsing since fast growth is generally associated with higher digestibility and lower defense against herbivores. However, studies linking browsing-induced trait variations to subsequent attractiveness to herbivores are still lacking, especially in the forest context. In this study, we experimentally examine the existence of a feedback loop between oak ( Quercus robur L.) and roe deer ( Capreolus capreolus ) and investigate its underlying morphological and chemical traits. We simulated single and repeated roe deer browsing on nursery-grown oak saplings and measured the changes in sapling height growth, lateral branching, leaf traits and winter shoot traits over two years. We conducted winter feeding trials with tame roe deer one year after the first treatment to test the effect of simulated browsing on sapling attractiveness. Simulated browsing reduced sapling height growth but had no effect on branching. Simulated browsing had no effect on leaf traits after half a year, but decreased the phenolic content and increased the fiber content of winter shoots the following winter. Contrary to our predictions, roe deer preferentially browsed control saplings over saplings previously browsed. After two years, repeated browsing promoted fast carbon acquisition leaf traits (high chlorophyll, high specific leaf area and low fiber content), reduced leaf phenolic content and increased leaf digestibility. We showed that a reduction in 1-year-old oak sapling height growth following browsing, combined with increased structural defense at the expense of chemical defense in winter shoots the following winter, was correlated with reduced browsing pressure, thereby challenging the feedback loop hypothesis. However, we also demonstrated that repeated browsing promoted fast carbon acquisition leaf traits in 2.5-year-old saplings, which tend to support the existence of a feedback loop on older and more intensively browsed saplings. As such, our study provides empirical evidence that morphological and physiological trait responses to browsing influence oak sapling attractiveness, but that the direction and magnitude of this effect depend on the ontogenic stage of the sapling and on the number of browsing events. • We examine the morphological and chemical response of oak saplings to browsing. • Browsing reduces sapling height growth and modifies shoots chemical composition. • We show with feeding trials that roe deer prefers unbrowsed saplings. • Oak trait responses to browsing decrease the probability of subsequent herbivory.

Specialized compounds across ontogeny in the seagrass Posidonia oceanica

Differences in phenolic composition across different ontogenic stages can be crucial in determining the interaction outcomes between plants and their surrounding biotic environment. In seagrasses, specific phenolic compounds have rarely been analyzed and remain unexplored in ontogenic stages other than non-reproductive adults. Furthermore, it is generally accepted that plants would prioritize defense (e.g., through increased phenolic content) on tissues or stages that are critical for plant fitness but how this affects nutritional quality or plant resources has been scarcely explored. We analyzed how phenolic composition, N and C content and carbohydrate resources varied among different life stages (i.e. old and young leaves of reproductive and non-reproductive plants, and leaves of seedlings) in the seagrass Posidonia oceanica. We identified five phenolic compounds, whose structures were established as hydroxycinnamate esters of tartaric acid. Also, our results show that in all examined ontogenic stages phenolic compounds have the same qualitative composition but inflorescences exhibit higher contents than vegetative tissues. We did not find a reduction in stored resources in reproductive plants, pointing to some kind of compensatory mechanism in the production or storage of resources. In contrast, seedlings seemed to have less phenolic compounds than reproductive plants, perhaps due to limited resources available to allocate to phenolic production. Our results demonstrate how different ontogenic stages change their investment in specialized phenolic compounds prioritizing different functions according to the needs and limitations of that stage.

TAPHONOMY AND DEPOSITIONAL SETTING OF THE SHRINGASAURUS INDICUS (ARCHOSAUROMORPHA: ALLOKOTOSAURIA) BONEBED FROM THE MIDDLE TRIASSIC DENWA FORMATION, SATPURA GONDWANA BASIN, INDIA

ABSTRACT A bonebed of multiple skeletons of the Triassic horned reptile Shringasaurus indicus was discovered in the upper Denwa Formation, Satpura Gondwana Basin, India. The monotaxic bonebed contains multiple individuals of different ontogenic stages indicating herding behavior by Shringasaurus indicus. The herd was a mixed-sex herd. The adult and sub-adult bones in the bonebed exceed the number of juvenile bones. The distribution of the bones was slightly patchy, bones of different individuals were admixed, and several bones were piled up implying mass mortality. The bonebed occurs in a fine-grained mudrock that is hydraulically incompatible with long-distance transport and concentration by currents. Sedimentary facies analysis indicates that the bonebed accumulated and was buried in a crevasse splay deposit between two ENE-WSW trending channel-fill complexes. The northern channel-fill complex was formed by unidirectional flow with lateral channel migration towards the south and with minor contemporaneous tectonic subsidence. Repeated breaching of the levee by this channel flow led to the incremental development of the crevasse splay deposit. The herd of Shringasaurus indicus, which lived near to the perennial channel, was drowned en masse and the carcasses were trapped within the muddy sediments of the crevasse splay deposit. Apart from a partially articulated skeleton, the rest of the bones were disarticulated but remained associated. The bones show little evidence of post-mortem modifications. With a continuous supply of the sediments through the spillover channels, the bones were buried before complete disarticulation and dispersal had taken place.

Effects of climate variability on an estuarine green crab Carcinus maenas population

The increase in frequency and intensity of extreme climate events over the last few decades has been leading to profound changes in estuarine and marine ecosystems worldwide, with strong implications for the species inhabiting these ecosystems as well as for the services provided by them. In this study, we analysed the effects of climate variability on the temporal and spatial variations in population dynamics of the green crab Carcinus maenas in the Mondego estuary (Portugal), between 2003 and 2018. In this 15-year period, a greater recruitment of C. maenas was observed during drought periods, periods which was matched by an increase in secondary production. Ontogenic stage segregation was also observed, with juveniles being found mainly in the further upriver areas of the estuary. The estuarine population was mainly composed of the green morphotype, with the orange and red morphotypes present in more downstream areas of the estuary. Redundancy analysis (RDA) showed high spatial and temporal variability of C. maenas in the estuary which was related with environmental changes over the 15-year period. A correlation between C. maenas biological features and several local-scale (salinity and river runoff) and large-scale (North Atlantic Oscillation index and Eastern Atlantic pattern) environmental variables was identified through cumulative sums analysis (CUSUM), indicating a strong environmental control on C. maenas population dynamics. This paper shows the importance of relatively long-term datasets to unravel the effects of extreme weather events due to climate change on key epibenthic estuarine species, and also how they might cope with a changing marine environment.

Landscape management among the Guarani of the Atlantic Forest of Misiones, Argentina: the case of the Syagrus romanzoffiana (Cham.) Glassman (Arecaceae) palm tree

The study of the management practices that different Guarani settlements have carried out in the environment over time constitutes one of the objectives of ethnoecology and historical ecology as the way through which these communities obtain forest resources affects landscape settings. In this work, based on a local classification system of the ontogenic stages of the pindo palm tree Syagrus romanzoffiana (Cham.) Glassman (Arecaceae), we study the effects of traditional management practices on the unity of the pindoty landscape - plant communities with a high concentration of this species. We evaluated the degree to which two morphometric variables used in the measurements of woody tree species, diameter at chest height (DBH) and total height (H), explain the local classification. As a result, in the environment of four Guarani settlements (Ita Piru, Kurupayty, Pindo Poty, and Yvyra Pepe Poty), the production of edible larvae favors temporary changes in the pindoty landscape unit since it produces changes in pindo population’s structure – (abundances by class size). In addition, we discuss some aspects of the pindoty units and their anthropogenic nature concluding that their evolution results from the Guarani cyclical cosmological conception.

Polyphagous caterpillars of Spodoptera litura switch from a trap crop to the main crop, improve fitness, and shorten generation time

Trap crops are used for pulling the pest load from the main crops toward themselves. Here, we report a switching by a polyphagous pest, Spodoptera litura, from trap crop castor to main crop eggplant during its mid-larval development. In the eggplant–castor mixed field, adults oviposit exclusively on castor. However, 3rd and 4th instar castor-grown larvae migrate to eggplants. Host choice assays for adults and larvae conducted in the controlled environment and the nutritional indices of castor and eggplant-feeding larvae revealed that the natural enemies did not influence the host switch. The host switch was rather associated with larvae’s enhanced digestion and metabolism. It shortened the development time and increased the eclosion success on eggplant. Mother-selected castor is suitable for the early instars and eggplant is for the later instars, indicating that the host switching was not because of the mother’s erroneous host choice but has evolved as an advantageous behavior. S. litura’s ability to switch hosts in the same ontogenic phase and reduce the generation time can contribute to an additional generation within a cropping season and endanger the critical growth stage of fruiting. Our findings support the ‘mother knows the best’ concept and annex that the larvae can also possess the ability to know the best. Such ability in different ontogenic stages can be fundamental to the exceptional adaptability of species like S. litura.

A Molecular Approach for the Detection and Quantification of Tribolium castaneum (Herbst) Infestation in Stored Wheat Flour.

SUMMARYResearch backgroundThe presence of insect fragments is one of the major constrains in stored food commodities and it causes considerable loss in the quality of the produce. The management of the pest is viewed as a huge challenge in foodprocessingindustry. Conventionally, the detection of Tribolium castenaum in the food processing industry is carried out by acid hydrolysis and staining methods that are time consuming and lack precision.Experimental approachConsidering the importance of a quick and effective method, a quantitative polymerase chain reaction (qPCR)-based approach was developed and elucidated in this study. The mitochondrial cytochrome oxidase I (mtCOI) gene was identified as a target due to its abundance in the pest. Specific primers were designed against the target gene by Primer Premier software and amplified in a qPCR.Results and conclusionsThis method is capable of detecting all the ontogenic stages of T. castaneum in stored wheat flour. Earlier experiments had demonstrated that about 20 µg of DNA can be obtained from 2.2 mg of insects. To quantify the infestation levels, the cycle threshold (Ct) values obtained from known samples were subjected to regression analysis and expressed as adult equivalents. In the unknown samples, the infestation was calculated as 1.74 and 0.046 adult insects in 5 g of wheat flour. The maximum permissible limit of insect fragments in flour is 75 insect fragments or approx. 3 adults per 50 g of flour as per the US Food and Drug Administration (FDA). Hence, by adopting this new method, it is possible for the warehouse operators to arrive at a decision to proceed with efficient management practices where wheat flour is stored. Also, this method can be ratified by government agencies associated with international business to ascertain whether the wheat flour meets the standards set by the respective country before subjecting to foreign trade.Novelty and scientific contributionThis study is the first of its kind in the detection and quantification of T. castaneum in milled products. So far, only conventional methods have been employed to assess the presence of the pests and manual counting of fragments are practiced to quantify the infestation levels. The developed qPCR method is faster, reliable and can be employed in milling industries, bakery industries, food processing plants and foreign trade units for critical detection and quantification of T. castaneum pest infestation.

Expression of immune-related genes during early development of the scallop Argopecten purpuratus after Vibrio splendidus challenge

Massive mortalities in hatchery-reared larvae of Argopecten purpuratus scallop are recurrent and have been associated regularly with Vibrio bacterial infections. Few studies have explored the immune system during the early development of scallops. To gain new insights on this subject, the expression patterns of four genes potentially involved in different phases of A. purpuratus immune response (sensing, signaling, and effectors) were assessed at different larval stages (trochophore, D-veliger, veliger, and pediveliger) and early juveniles from fall and spring spawning seasons. A Toll-like receptor (ApTLR), an inhibitor of nuclear factor kappa B (ApIκB), and two antimicrobial effectors (big defensin ApBD1 and ferritin Apfer1) were measured at each developmental stage. Gene expression was determined from control and challenged pools with V. splendidus VPAP18 (pathogenic strain) after 6, 15, and 24 h. Results revealed that expression patterns of all genes were consistent between the assessed spawning seasons. Regardless of the immune status, transcriptional levels of ApTLR, ApBD1, and Apfer1 gradually increased during ontogeny. ApIkB displayed a contrasted transcriptional pattern, with higher expression in the early ontogenic stages, suggesting its participation in other metabolic processes at earlier stages. Post-Vibrio challenge overexpression was observed mainly in the pediveligers and early juveniles for ApTLR and antimicrobial effectors, with ApBD1 showing the strongest and consistent response. Thus, ApBD1 protein availability was assessed by immunofluorescence. ApBD1 was present on every developmental stage (control and Vibrio challenged), but at a higher level in challenged pediveligers. Overall, results suggest a maturation of the immune system towards the late developmental stages (pediveligers and early juveniles) and a possible parental transfer of ApBD1. The present study contributes to scallop's early ontogeny immunobiology knowledge and for the establishment of the management disease process in hatcheries of A. purpuratus.

Not only greening: The effects of marennine produced by Haslea ostrearia on physiological traits of three bivalve species

The marine diatom Haslea ostrearia is known for its ability to produce marennine, a blue-green water-soluble pigment, which is responsible for the oyster greening phenomenon in the French Atlantic Coast. Marennine can be considered a bioactive extracellular compound (BEC) as it exhibits activities against detrimental pathogens in bivalve aquaculture. However, marennine could also be toxic to animals. The aim of this experimental study was to assess the effect of marennine produced by H. ostrearia upon three commercially important bivalve species, the American oyster Crassostrea virginica, the blue mussel Mytilus edulis and the giant scallop Placopecten magellanicus. Effect of this BEC was also assessed on different ontogenic stages of scallops. The animals were exposed to 3 mg L−1 of marennine and physiological response associated with clearance rates (CR), oxygen consumption and condition index (CI) were measured after 16 h of exposure (day 0), and after one subsequent week of recovery (day 7). In the short term, CR and oxygen consumption on mussels and oysters were significantly altered by marennine but not for scallops. CR recovery was observed in oysters after seven days, but no depuration was observed in mussels. Nevertheless, oxygen consumption in both mussels and oysters recovered on day 7. CI in the marennine-exposed group for both adult oysters and scallops was not significantly different, yet CI of mussels was lowered by 18.2 % compared to control. Scallop adults seemed largely resistant to marennine, but juveniles were more susceptible with a reduced CR and oxygen consumption by 71.9 % and 24.5 %, respectively, and no depuration activity observed until day 7. Additionally, CI in marennine-exposed juvenile scallops was 27 % lower than that of control. This study demonstrates species- and age-specific effect of marennine on bivalves, thus highlighting the importance of further assessment on the utilization of this pigment as a natural product decreasing pathogenicity of bacteria in shellfish aquaculture systems.

Characterization of Insulin-Like Growth Factor Binding Protein-5 (IGFBP-5) Gene and Its Potential Roles in Ontogenesis in the Pacific Abalone, Haliotis discus hannai.

Insulin-like growth factor binding protein family is known to be involved in regulating biological actions of insulin-like growth factors (IGFs). In the present study, a full-length cDNA encoding the IGFBP-5 gene was cloned and characterized from the cerebral ganglion of Haliotis discus hannai. The 921-bp full-length sequence of Hdh IGFBP-5 cDNA transcript had an open reading frame of 411 bp encoding a predicted polypeptide of 136 amino acids, sharing high sequence identities with IGFBP-5 of H. diversicolor. The deduced Hdh IGFBP-5 protein contained a putative transmembrane domain (13-35 aa) in the N-terminal region. It also possessed a signature domain of IGFBP protein family (IB domain, 45-120 aa). Six cysteine residues (Cys-47, Cys-55, Cys-73, Cys-85, Cys-98, and Cys-118) in this cloned sequence could potentially form an intrachain disulfide bond. Phylogenetic analysis indicated that the Hdh IGFBP-5 gene was robustly clustered with IGFBP-5 of H. diversicolor. Tissue distribution analysis based on qPCR assay showed that Hdh IGFBP-5 was widely expressed in all examined tissues, with significantly (p < 0.05) higher expression in the cerebral ganglion. In male and female gametogenetic cycles, Hdh IGFBP-5 mRNA was expressed at all stages, showing significantly higher level at ripening stage. The expression level of Hdh IGFBP-5 mRNA was significantly higher in the polar body stage than in other ontogenic stages. In situ hybridization revealed that Hdh IGFBP-5 mRNA was present in the neurosecretory cells of the cerebral ganglion. This is the first study describing IGFBP-5 in H. discus hannai that might be synthesized in the neural ganglia. Our results demonstrate Hdh IGFBP-5 is involved in regulating ontogenic development and reproductive regulation of H. discus hannai.

Contrasting species decline but high sensitivity to increasing water stress on a mixed pine–oak ecotone

Abstract Forest decline under environmental stress is expressed by regeneration failure and accelerated mortality in all ontogenic stages at the population level. Characterizing functional traits and mechanisms that best capture species decline and mortality is essential to assess forest dynamics. We analysed sensitivity to increasing water stress in two species with different water‐use strategies on a mixed Quercus pyrenaica–Pinus sylvestris forest where adult pines express vulnerability to climate change but oaks do not. We compared the dynamics of radial growth, wood δ13C and sapwood non‐structural carbohydrates (NSCs) in response to drought at different time‐scales in both species and two age cohorts in pine. Both species were very sensitive to water stress, which influenced trait phenotypic plasticity at short‐ and long time‐scales. Water‐use strategy in pines of both ages was more conservative than in the more drought‐tolerant oak. Both species showed negative growth trends despite increasing intrinsic water‐use efficiency. Recent growth of pines is slower than it was in the past. Carbon isotope discrimination trends in young pines suggested increasing leaf gas exchange constraints. NSCs were far from depletion in both species and all pine ages. Intra‐ and inter‐annual NSC variability was higher in oaks than in pines and in soluble sugars (SS) than in starch. SS were lowest in young pines. Sensitivity of NSCs to contrasting climatic years was low in pines, and NSC levels mostly remained homeostatic for this species. The sensitivity to climate expressed suggests different C allocation strategies, with less coupling between radial growth and current‐year photosynthesis in young pines. Synthesis. Pines expressed negative responses to increased water stress regardless of age, showing rising gas exchange constraints through tighter stomatal control of water losses than oaks. Young pines showed similar functional responses to water stress than old pines in decline, which suggests species‐level vulnerability and could be regarded as early warning signals anticipating mortality in pines. Yet, given the high sensitivity to drought also expressed by the non‐declining oak, it would have been difficult to unequivocally disentangle species decline based only on the functional traits analysed.