Exploring the co-operativity of secretory structures for defense and pollination in flowering plants.

MAIN CONCLUSION: In flowers multiple secretory systems cooperate to deliver specialized metabolites to support specific roles in defence and pollination. The collective roles of cell types, enzymes, and transporters are discussed. The interplay between reproductive strategies and defense mechanisms in flowering plants has long been recognized, with trade-offs between investment in defense and reproduction predicted. Glandular trichomes and secretory cavities or ducts, which are epidermal and internal structures, play a pivotal role in the secretion, accumulation, and transport of specialized secondary metabolites, and contribute significantly to defense and pollination. Recent investigations have revealed an intricate connection between these two structures, whereby specialized volatile and non-volatile metabolites are exchanged, collectively shaping their respective ecological functions. However, a comprehensive understanding of this profound integration remains largely elusive. In this review, we explore the secretory systems and associated secondary metabolism primarily in Asteraceous species to propose potential shared mechanisms facilitating the directional translocation of these metabolites to diverse destinations. We summarize recent advances in our understanding of the cooperativity between epidermal and internal secretory structures in the biosynthesis, secretion, accumulation, and emission of terpenes, providing specific well-documented examples from pyrethrum (Tanacetum cinerariifolium). Pyrethrum is renowned for its natural pyrethrin insecticides, which accumulate in the flower head, and more recently, for emitting an aphid alarm pheromone. These examples highlight the diverse specializations of secondary metabolism in pyrethrum and raise intriguing questions regarding the regulation of production and translocation of these compounds within and between its various epidermal and internal secretory systems, spanning multiple tissues, to serve distinct ecological purposes. By discussing the cooperative nature of secretory structures in flowering plants, this review sheds light on the intricate mechanisms underlying the ecological roles of terpenes in defense and pollination.

Understanding pyrethrin biosynthesis: toward and beyond natural pesticide overproduction.

Pyrethrins are natural insecticides biosynthesised by Asteraceae plants, such as Tanacetum cinerariifolium and have a long history, dating back to ancient times. Pyrethrins are often used as low-persistence and safe insecticides to control household, horticultural, and agricultural insect pests. Despite its long history of use, pyrethrin biosynthesis remains a mystery, presenting a significant opportunity to improve yields and meet the growing demand for organic agriculture. To achieve this, both genetic modification and non-genetic methods, such as chemical activation and priming, are indispensable. Plants use pyrethrins as a defence against herbivores, but pyrethrin biosynthesis pathways are shared with plant hormones and signal molecules. Hence, the insight that pyrethrins may play broader roles than those traditionally expected is invaluable to advance the basic and applied sciences of pyrethrins.

Nocturnal burst emissions of germacrene D from the open disk florets of pyrethrum flowers induce moths to oviposit on a nonhost and improve pollination success.

Recent studies show that nocturnal pollinators may be more important to ecosystem function and food production than is currently appreciated. Here, we describe an agricultural field study of pyrethrum (Tanacetum cinerariifolium) flower pollination. Pyrethrum is genetically self-incompatible and thus is reliant on pollinators for seed set. Our pollinator exclusion experiment showed that nocturnal insects, particularly moths, significantly contribute to seed set and quality. We discovered that the most abundant floral volatile, the sesquiterpene (-)-germacrene D (GD), is key in attracting the noctuid moths Peridroma saucia and Helicoverpa armigera. Germacrene D synthase (GDS) gene expression regulates the specific GD production and accumulation in flowers, which, in contrast to related species, lose the habit of closing at night. We did observe that female moths also oviposited on pyrethrum leaves and flower peduncles, but found that only a small fraction of those eggs hatched. Larvae were severely stunted in development, most likely due to the presence of pyrethrin defense compounds. This example of exploitative mutualism, which blocks the reproductive success of the moth pollinator and depends on nocturnal interactions, is placed into an ecological context to explain why it may have developed.

Evolution of the Genetic Structure of the Didymella tanaceti Population During Development of Succinate Dehydrogenase Inhibitor Resistance.

Emergence of pathogens with decreased sensitivity to succinate dehydrogenase inhibitor fungicides is a global agronomical issue. Analysis of Didymella tanaceti isolates (n = 173), which cause tan spot of pyrethrum (Tanacetum cinerariifolium), collected prior to (2004 to 2005) and after (2009, 2010, 2012, and 2014) the commercial implementation of boscalid in Tasmanian pyrethrum fields identified that insensitivity developed over time and has become widespread. To evaluate temporal change, isolates were characterized for frequency of mutations in the succinate dehydrogenase (Sdh) B, C, and D subunits associated with boscalid resistance, mating type, and SSR genotype. All isolates from 2004 and 2005 exhibited wild-type (WT) Sdh alleles. Seven known Sdh substitutions were identified in isolates collected from 2009 to 2014. In 2009, 60.7% had Sdh substitutions associated with boscalid resistance in D. tanaceti. The frequency of WT isolates decreased over time, with no WT isolates identified in 2014. The frequency of the SdhB-H277Y genotype increased from 10.7 to 77.8% between 2009 and 2014. Genotypic evidence suggested that a shift in the population structure occurred between 2005 and 2009, with decreases in gene diversity (uh; 0.51 to 0.34), genotypic evenness (E5; 0.96 to 0.67), genotypic diversity (G; 9.3 to 6.8), and allele frequencies. No evidence was obtained to support the rapid spread of Sdh genotypes by clonal expansion of the population. Thus, insensitivity to boscalid has developed and become widespread within a diverse population within 4 years of usage. These results suggest that D. tanaceti can disperse insensitivity through repeated frequent mutation, sexual recombination, or a combination of both.

Phytochemical, Morphological and Genetic Characterisation of Anacyclus pyrethrum var. depressus (Ball.) Maire and Anacyclus pyrethrum var. pyrethrum (L.) Link.

The present study is based on a multidisciplinary approach carried out for the first time on Anacyclus pyrethrum var. pyrethrum and Anacyclus pyrethrum var. depressus, two varieties from the endemic and endangered medicinal species listed in the IUCN red list, Anacyclus pyrethrum (L.) Link. Therefore, morphological, phytochemical, and genetic characterisations were carried out in the present work. Morphological characterisation was established based on 23 qualitative and quantitative characters describing the vegetative and floral parts. The phytochemical compounds were determined by UHPLC. Genetic characterisation of extracted DNA was subjected to PCR using two sets of universal primers, rbcL a-f/rbcL a-R and rpocL1-2/rpocL1-4, followed by sequencing analysis using the Sanger method. The results revealed a significant difference between the two varieties studied. Furthermore, phytochemical analysis of the studied extracts revealed a quantitative and qualitative variation in the chemical profile, as well as the presence of interesting compounds, including new compounds that have never been reported in A. pyrethrum. The phylogenetic analysis of the DNA sequences indicated a similarity percentage of 91%. Based on the morphological characterisation and congruence with the phytochemical characterisation and molecular data, we can confirm that A. pyrethrum var. pyrethrum and A. pyrethrum var. depressus represent two different taxa.

Toxicological aspect of bioinsecticide pyrethrum extract and expressions of apoptotic gene levels in human hepotacellular carcinoma HepG2 cells.

Pyrethrum extract (PE), an important natural bioinsecticide, is extensively used across the world to control pest insects in homes and farms. The aim of this study was to evaluate the potential cytotoxic effect of PE using MTT assay and genotoxic effect using micronucleus (MN) assay. The changes in the expressions of the apoptosis genes in mRNA levels were also investigated using Real-Time qPCR analysis as well as the ratio of apoptotic/necrotic cells with AnnexinV-FITC/Propidium iodide (PI) assay in HepG2 cells. PE markedly suppressed the cell proliferation on HepG2 cells. It significantly increased the frequency of micronucleus (MN) at 500 and 1000 µg/mL. PE also induced the percentage of the cell population of late apoptotic/necrotic cells (FITC + PI+) and necrotic cells (FITC- PI+), especially at 4000 µg/mL analyzed by flow cytometry. PE caused significant fold changes in the expression of several apoptotic genes including APAF1, BIK, BAX, BAD, BID, MCL-1, CASP3, CASP1, CASP2, FAS, FADD and TNFRSF1A. In particular, the pro-apoptotic gene Hrk (Harakiri) remarkably and dose-dependently was overexpressed of the mRNA level. As a result, PE may exhibit cyto-genotoxic effects, especially at higher concentrations and lead to significant changes in the expression of mRNA levels in several apoptotic genes.HighlightsNatural bioinsecticide PE exhibited a cytotoxic effect in HepG2 cells.PE significantly induced the micronucleus (MN) frequency at 500 and 1000 µg/mL.This bioinsecticide induced cell death and it lead to significant fold changes in the expression of mRNA levels in several apoptotic genes in HepG2 cells.The highest increase of the expression of mRNA levels was determined in Hrk (Harakiri) at 4000 µg/mL.

Indoor and outdoor malaria transmission in two ecological settings in rural Mali: implications for vector control.

BACKGROUND: Implementation and upscale of effective malaria vector control strategies necessitates understanding the multi-factorial aspects of transmission patterns. The primary aims of this study are to determine the vector composition, biting rates, trophic preference, and the overall importance of distinguishing outdoor versus indoor malaria transmission through a study at two communities in rural Mali. METHODS: Mosquito collection was carried out between July 2012 and June 2016 at two rural Mali communities (Dangassa and Koïla Bamanan) using pyrethrum spray-catch and human landing catch approaches at both indoor and outdoor locations. Species of Anopheles gambiae complex were identified by polymerase chain reaction (PCR). Enzyme-Linked -Immuno-Sorbent Assay (ELISA) were used to determine the origin of mosquito blood meals and presence of Plasmodium falciparum sporozoite infections. RESULTS: A total of 11,237 An. gambiae sensu lato (s.l.) were collected during the study period (5239 and 5998 from the Dangassa and Koïla Bamanan sites, respectively). Of the 679 identified by PCR in Dangassa, Anopheles coluzzii was the predominant species with 91.4% of the catch followed by An. gambiae (8.0%) and Anopheles arabiensis (0.6%). At the same time in Koïla Bamanan, of the 623 An. gambiae s.l., An. coluzzii accounted for 99% of the catch, An. arabiensis 0.8% and An. gambiae 0.2%. Human Blood Index (HBI) measures were significantly higher in Dangassa (79.4%; 95% Bayesian credible interval (BCI) [77.4, 81.4]) than in Koïla Bamanan (15.9%; 95% BCI [14.7, 17.1]). The human biting rates were higher during the second half of the night at both sites. In Dangassa, the sporozoite rate was comparable between outdoor and indoor mosquito collections. For outdoor collections, the sporozoite positive rate was 3.6% (95% BCI [2.1-4.3]) and indoor collections were 3.1% (95% BCI [2.4-5.0]). In Koïla Bamanan, the sporozoite rate was higher indoors at 4.3% (95% BCI [2.7-6.3]) compared with outdoors at 2.4% (95% BCI [1.1-4.2]). In Dangassa, corrected entomological inoculation rates (cEIRs) using HBI were 13.74 [95% BCI 9.21-19.14] infective bites/person/month (ib/p/m) at indoor, and 18.66 [95% BCI 12.55-25.81] ib/p/m at outdoor. For Koïla Bamanan, cEIRs were 1.57 [95% BCI 2.34-2.72] ib/p/m and 0.94 [95% BCI 0.43-1.64] ib/p/m for indoor and outdoor, respectively. EIRs were significantly higher at the Dangassa site than the Koïla Bamanan site. CONCLUSION: The findings in this work may indicate the occurrence of active, outdoor residual malaria transmission is comparable to indoor transmission in some geographic settings. The high outdoor transmission patterns observed here highlight the need for additional strategies to combat outdoor malaria transmission to complement traditional indoor preventive approaches such as long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) which typically focus on resting mosquitoes.

Botanical Insecticides in the Twenty-First Century-Fulfilling Their Promise?

Academic interest in plant natural products with insecticidal properties has continued to grow in the past 20 years, while commercialization of new botanical insecticides and market expansion of existing botanicals has lagged considerably behind. Insecticides based on pyrethrum and neem (azadirachtin) continue to be standard bearers in this class of pesticides, but globally, their increased presence is largely a consequence of introduction into new jurisdictions. Insecticides based on plant essential oils are just beginning to emerge as useful plant protectants. Some countries (such as Turkey, Uruguay, the United Arab Emirates, and Australia) have relaxed regulatory requirements for specific plant extracts and oils, while in North America and the European Union, stricter requirements have slowed progress toward commercialization of new products. Botanicals are likely to remain niche products in many agricultural regions and may have the greatest impact in developing countries in tropical regions where the source plants are readily available and conventional products are both expensive and dangerous to users.

Anacyclus pyrethrum (L): Chemical Composition, Analgesic, Anti-Inflammatory, and Wound Healing Properties.

BACKGROUND: Anacyclus pyrethrum (A. pyrethrum) is a wild species belonging to the family Asteraceae, which is used in traditional medicines. AIM OF THE STUDY: This work was undertaken to study the chemical composition, analgesic, anti-inflammatory, and wound healing properties of hydroalcoholic extracts of different parts (roots, seeds, leaves, and capitula) of A. pyrethrum. Material and Methods: The phytochemical analysis of the studied extracts was conducted by GC-MS. The analgesic activity was evaluated in mice using acetic acid and formaldehyde methods. The anti-inflammatory activity was tested using the inhibitory method of edema induced in rats. The healing activity of the hydroethanolic extracts was explored by excision and incision wound healing models in rats. RESULTS: The phytochemical analysis of the studied plant extracts affirmed the presence of interesting compounds, including some newly detected elements, such as sarcosine, N-(trifluoroacetyl)-butyl ester, levulinic acid, malonic acid, palmitic acid, morphinan-6-One, 4,5.alpha.-epoxy-3-hydroxy-17-methyl, 2,4-undecadiene-8,10-diyne-N-tyramide, and isovaleric acid. The extracts of different parts (roots, seeds, leaves, and capitula) exhibited promising anti-inflammatory, analgesic, and wound healing effects, with percentages of inhibition up to 98%, 94%, and 100%, respectively. CONCLUSION: This study might contribute towards the well-being of society as it provides evidence on the potential analgesic, anti-inflammatory, and wound healing properties of A. pyrethrum.

Population Structure of Colletotrichum tanaceti in Australian Pyrethrum Reveals High Evolutionary Potential.

Colletotrichum tanaceti, the causal agent of anthracnose, is an emerging pathogen of commercially grown pyrethrum (Tanacetum cinerariifolium) in Australia. A microsatellite marker library was developed to understand the spatio-genetic structure over three sampled years and across two regions where pyrethrum is cultivated in Australia. Results indicated that C. tanaceti was highly diverse with a mixed reproductive mode; comprising both sexual and clonal reproduction. Sexual reproduction of C. tanaceti was more prevalent in Tasmania than in Victoria. Little differentiation was observed among field populations likely due to isolation by colonization but most of the genetic variation was occurring within populations. C. tanaceti was likely to have had a long-distance gene and genotype flow among distant populations within a state and between states. Anthropogenic transmission of propagules and wind dispersal of ascospores are the most probable mechanisms of long-distance dispersal of C. tanaceti. Evaluation of putative population histories suggested that C. tanaceti most likely originated in Tasmania and expanded from an unidentified host onto pyrethrum. Victoria was later invaded by the Tasmanian population. With the mixed mode of reproduction and possible long-distance gene flow, C. tanaceti is likely to have a high evolutionary potential and thereby has ability to adapt to management practices in the future.

Action of six pyrethrins purified from the botanical insecticide pyrethrum on cockroach sodium channels expressed in Xenopus oocytes.

Pyrethrin I, pyrethrin II, cinerin I, cinerin II, jasmolin I and jasmolin II are six closely related insecticidal active esters, known as pyrethrins, found in the pyrethrum extract from the dry flowers of Tanacetum cinerariifolium. The chemical structures of the six compounds differ only in the terminal moieties at the acid and alcohol ends, but the compounds' in vivo toxicities are substantially different. Pyrethrins are lead compounds for pyrethroids, a large family of synthetic insecticides that alter nerve functions by prolonging the opening of voltage-gated sodium channels. However, data on the mechanism of action of natural pyrethrins are very limited. In this study, we examined the actions of all six pyrethrins on cockroach sodium channels expressed in Xenopus oocytes. Although the six compounds showed comparable potencies in inhibiting the inactivation of sodium channels, they had greatly variable potencies in inhibiting channel deactivation. Furthermore, unlike pyrethroids, the action of pyrethrins neither depend on nor were enhanced by repeated channel activation. We created a NavMs-based model of the cockroach sodium channel, in which pyrethrin II was docked at the pyrethroid receptor site 1 (PyR1), and proposed a rationale for the observed structure-activity relationship of the six pyrethrins. Our study sheds light on the molecular mechanism of pyrethrum action on sodium channels and reveled differences in the modes of action of the six bioactive constitutes of pyrethrum.

Potential of pyrethroid-synergised pyrethrum on stored product insects and implications for use as prophylactic sprays.

The study was conducted to evaluate the synergistic effects of 2% pyrethrum extract with synthetic pyrethroids on the mortality of stored product insects. Contact toxicity was performed at variable concentrations observing mortality at 12, 24 and 48 h durations. The results of the present study indicated that, pyrethrum + deltamethrin combination (25:1 ratio) was effective on the adults of Rhyzopertha dominica (F.) and Tribolium castaneum (Herbst). On the other hand, pyrethrum + cypermethrin combination proved effective against Sitophilus oryzae (L.). The efficacy of the tested combination showed reasonable increase in mortality response in treated insects over increasing exposures. At 48 h, 450 ppm pyrethrum + deltamethrin combination induced 25, 90 and 97% mortalities in S. oryzae, T. castaneum and R. dominica adults; while, pyrethrum-cypermethrin combination recorded 75, 45 and 75% mortalities respectively. On the other hand, it was observed that, among the pyrethrum alone treatments i.e. at 300, 450 and 600 ppm concentrations, maximum mortality (62.5%) was observed in S. oryzae exposed to 600 ppm pyrethrum for 48 h. The effective LC50 concentrations for pyrethrum (600 ppm) + deltamethrin combination was estimated to be as 0.1987 and 0.7039 µl/cm2 for R. dominica and T. castaneum adults respectively. Contrastingly, for treatments with S. oryzae, a LC50 value of 0.8673 µl/cm2 was recorded for pyrethrum (600 ppm) + cypermethrin mixture. This investigation strengthens the fact that pyrethrum along with pyrethroids is effective against storage insect pests which can be promisingly a safer insecticidal combination.

Pyrethrum-extract induced autophagy in insect cells: A new target?

Pyrethrum extract (PY) is a natural insecticide that is extensively used across the world, and its insecticidal activity is attributed to the presence of six active esters known as pyrethrins. PY targets the nervous systems of insects by delaying the closure of voltage-gated sodium ion channels in the nerve cells. However, limited information is available regarding the toxicity and detailed mechanisms of PY activity. This study is aimed at understanding the toxicity effect and the underlying mechanisms of PY in cellular level, which have not yet been investigated on the non-nervous system of insects. Results of the MTT assay showed that the viability of Sf9 cells was inhibited by PY in a time- and concentration-dependent manner, and observation under a microscope revealed accumulation of intracellular vacuoles. Monodansylcadaverine staining analysis and transmission electron microscope images revealed typical autophagic morphological changes in PY-treated Sf9 cells. Autophagy-related proteins such as LC3, p62, and beclin-1 were detected using by Western blotting. Protein expression levels of LC3-II and beclin-1 were upregulated while that of p62 was markedly downregulated in a dose-dependent manner upon the PY treatment in Sf9 cells. In conclusion, these results indicate that PY could induce autophagy in the non-nervous system of insects which may contribute to its insecticidal mechanism.

Alkamides from Anacyclus pyrethrum L. and Their in Vitro Antiprotozoal Activity.

In our ongoing study to evaluate the antiprotozoal activity of alkamides from Asteraceae, a dichloromethane extract from the roots of Anacycluspyrethrum L. showed a moderate in vitro activity against the NF54 strain of Plasmodium falciparum and against Leishmaniadonovani (amastigotes, MHOM/ET/67/L82 strain). Seven pure alkamides and a mixture of two further alkamides were isolated by column chromatography followed by preparative high performance liquid chromatography. The alkamides were identified by mass- and NMR-spectroscopic methods as tetradeca-2E,4E-dien-8,10-diynoic acid isobutylamide (anacycline, 1), deca-2E,4E-dienoic acid isobutylamide (pellitorine, 2), deca-2E,4E,9-trienoic acid isobutylamide (3), deca-2E,4E-dienoic acid 2-phenylethylamide (4), undeca-2E,4E-dien-8,10-diynoic acid isopentylamide (5), tetradeca-2E,4E,12Z-trien-8,10-diynoic acid isobutylamide (6), and dodeca-2E,4E-dien acid 4-hydroxy-2-phenylethylamide (7). Two compounds-undeca-2E,4E-dien-8,10-diynoic acid 2-phenylethylamide (8) and deca-2E,4E-dienoic acid 4-hydroxy-2-phenylethylamide (9)-were isolated as an inseparable mixture (1:4). Compounds 3, 4, and 5 were isolated from Anacycluspyrethrum L. for the first time. While compounds 4 and 5 were previously known from the genus Achillea, compound 3 is a new natural product, to the best of our knowledge. All isolated alkamides were tested in vitro for antiprotozoal activity against Plasmodium falciparum, Trypanosomabruceirhodesiense, Trypanosomacruzi, and Leishmaniadonovani and for cytotoxicity against L6 rat skeletal myoblasts.

[Study on screening antitumor active fractions and chemical components in active fractions from root of Anaycclus pyrethrum].

Components that systematic separated from the root of Anaycclus pyrethrum were identified, in order to lay a foundation for future study of the root of A. pyrethrum. The CCK-8 assay showed that dichloromethane fraction exhibited the highest degree of cytotoxicity than others. Ten monomeric components were obtained from dichloromethane fraction and ethyl acetate fraction extracted from the root of A. pyrethrum, including 7 N-alkylamides, one coumarin and two flavonoid glycosides. They were identified as tetradeca-2E,4E,8E-trienoic acid 4-hydroxyphenylethylamide(1), deca-2E,4E-dienoicacid isobutylamide(2), undeca-2E,4E-diene-8,10-diynoic acid phenylethylamide(3), tetradeca-2E,4E-dienoic acid 4-hydroxyphenylethylamide(4), tetradeca-2E,4E-diene-8,10-diynoic acid isobutylamide(5), deca-2E,4E- dienoic acid 4-hydroxyphenylethylamide(6), dodeca-2E,4E-dienoic acid 4-hydroxy -phenyl-ethylamide(7), isoscopoletin(8), quercetin-7-O-ß-D-glucopyranoside(9), isorhamnetin-7-O-ß-D-glucopyranoside(10). Among them, compound 1 was identified as a new compound, Compounds 2-4, 8-10 were isolated from this herb for the first time.

Conifer flavonoid compounds inhibit detoxification enzymes and synergize insecticides.

Detoxification by glutathione S-transferases (GSTs) and esterases are important mechanisms associated with insecticide resistance. Discovery of novel GST and esterase inhibitors from phytochemicals could provide potential new insecticide synergists. Conifer tree species contain flavonoids, such as taxifolin, that inhibit in vitro GST activity. The objectives were to test the relative effectiveness of taxifolin as an enzyme inhibitor and as an insecticide synergist in combination with the organophosphorous insecticide, Guthion (50% azinphos-methyl), and the botanical insecticide, pyrethrum, using an insecticide-resistant Colorado potato beetle (CPB) Leptinotarsa decemlineata (Say) strain. Both taxifolin and its isomer, quercetin, increased the mortality of 1(st) instar CPB larvae after 48h when combined with Guthion, but not pyrethrum. Taxifolin had greater in vitro esterase inhibition compared with the commonly used esterase inhibitor, S, S, S-tributyl phosphorotrithioate (DEF). An in vivo esterase and GST inhibition effect after ingestion of taxifolin was measured, however DEF caused a greater suppression of esterase activity. This study demonstrated that flavonoid compounds have both in vitro and in vivo esterase inhibition, which is likely responsible for the insecticide synergism observed in insecticide-resistant CPB.

Biases encountered in long-term monitoring studies of invertebrates and microflora: Australian examples of protocols, personnel, tools and site location.

Monitoring forms the basis for understanding ecological change. It relies on repeatability of methods to ensure detected changes accurately reflect the effect of environmental drivers. However, operator bias can influence the repeatability of field and laboratory work. We tested this for invertebrates and diatoms in three trials: (1) two operators swept invertebrates from heath vegetation, (2) four operators picked invertebrates from pyrethrum knockdown samples from tree trunk and (3) diatom identifications by eight operators in three laboratories. In each trial, operators were working simultaneously and their training in the field and laboratory was identical. No variation in catch efficiency was found between the two operators of differing experience using a random number of net sweeps to catch invertebrates when sequence, location and size of sweeps were random. Number of individuals and higher taxa collected by four operators from tree trunks varied significantly between operators and with their 'experience ranking'. Diatom identifications made by eight operators were clustered together according to which of three laboratories they belonged. These three tests demonstrated significant potential bias of operators in both field and laboratory. This is the first documented case demonstrating the significant influence of observer bias on results from invertebrate field-based studies. Examples of two long-term trials are also given that illustrate further operator bias. Our results suggest that long-term ecological studies using invertebrates need to be rigorously audited to ensure that operator bias is accounted for during analysis and interpretation. Further, taxonomic harmonisation remains an important step in merging field and laboratory data collected by different operators.

Antagonistic and additive effect when combining biopesticides against the fall armyworm, Spodoptera frugiperda.

Fall armyworm, Spodoptera frugiperda (FAW) is a cosmopolitan crop pest species that has recently become established in sub-Saharan Africa and Southeast Asia. Current FAW control is almost entirely dependent on synthetic pesticides. Biopesticides offer a more sustainable alternative but have limitations. For example, pyrethrum is an effective botanical insecticide with low mammalian toxicity but is highly UV labile, resulting in a rapid loss of efficacy in the field. Beauveria bassiana is an entomopathogenic fungus that is more persistent, but there is a time lag of several days before it causes insect mortality and leads to effective control. The combination of these biopesticides could mitigate their drawbacks for FAW control. Here we evaluated the efficacy of pyrethrum and B. bassiana as individual treatments and in combination against 3rd instar FAW. Four different combinations of these two biopesticides were tested, resulting in an antagonistic relationship at the lowest concentrations of B. bassiana and pyrethrum (1 × 104 conidia mL-1 with 25 ppm) and an additive effect for the other 3 combined treatments (1 × 104 conidia mL-1 with 100 ppm and 1 × 105 conidia mL-1 with 25 ppm and 100 ppm pyrethrum). Additionally, a delay in efficacy from B. bassiana was observed when combined with pyrethrum as well as a general inhibition of growth on agar plates. These results appear to show that this particular combination of biopesticides is not universally beneficial or detrimental to pest control strategies and is dependent on the doses of each biopesticide applied. However, the additive effect shown here at specific concentrations does indicate that combining biopesticides could help overcome the challenges of persistence seen in botanical pesticides and the slow establishment of EPF, with the potential to improve effectiveness of biopesticides for IPM.

Alkaloid constituents from Anacyclus pyrethrum.

Twelve undescribed alkaloids, including eight pyrrolo[3,2-g]isoquinoline alkaloids (+)/(-)-anacyquinoline A (1a/1b), (±)-anacyquinoline B (2), (+)/(-)-anacyquinoline C (3a/3b), (±)-anacyquinoline D (4), (±)-anacyquinoline E (5), and (±)-anacyquinoline F (6), together with four pyrrolo[2,3-g]quinoline alkaloids (+)/(-)-anacyquinoline G (7a/7b), (±)-anacyquinoline H (8), and (±)-anacyquinoline I (9), were isolated from the root of Anacyclus pyrethrum (L.) DC. Their structures were determined via spectroscopic analyses (UV, IR, NMR), HRESIMS, quantum chemical calculations of ECD and NMR data, and single-crystal X-ray diffraction analysis (Cu Kα). In bioassay, (+)/(-)-anacyquinoline G (7a/7b), and (±)-anacyquinoline H (8) showed inhibition on NO production with IC50 values of 41.4, 44.1, and 31.4 µM, respectively.

Alkaloids from Anacyclus pyrethrum.

Twelve undescribed alkaloids, including eight pyrrolo[3,2-g]isoquinoline alkaloids (+)/(-)-anacyquinoline A (1a/1b), (±)-anacyquinoline B (2), (+)/(-)-anacyquinoline C (3a/3b), (±)-anacyquinoline D (4), (±)-anacyquinoline E (5), and (±)-anacyquinoline F (6), together with four pyrrolo[2,3-g]quinoline alkaloids (+)/(-)-anacyquinoline G (7a/7b), (±)-anacyquinoline H (8), and (±)-anacyquinoline I (9), were isolated from the roots of Anacyclus pyrethrum (L.) DC. Their structures were determined via spectroscopic analyses (UV, IR, NMR), HRESIMS, quantum chemical calculations of ECD, DP4+ analysis, and single-crystal X-ray diffraction analysis (Cu Kα). Furthermore, in bioassay, (+)/(-)-anacyquinoline G (7a/7b) and (±)-anacyquinoline H (8) showed inhibition on nitric oxide production in lipopolysaccharide -induced RAW 264.7 cells with IC50 values of 41.4, 44.1, and 31.4 µM, respectively, indicating their potential anti-inflammatory bioactivity.