Insect Parasitoids Research Articles

Know When You Are Too Many: Density-Dependent Release of Pheromones During Host Colonisation by the European Spruce Bark Beetle, Ips typographus (L.)

Individuals across various animal species communicate their presence to conspecifics. Especially phytophagous and parasitoid insects with their brood developing on limited resources rely on chemical cues, such as host-marking pheromones, to reduce intraspecific competition. Bark beetles are phytophagous insects with some species being economically and ecologically relevant forest pests. Several of them use the volatile compound verbenone to inhibit attraction and reduce intraspecific competition. However, in the Eurasian spruce bark beetle, Ips typographus (L.), temporal emission patterns did so far not quite support the putative function of verbenone as an indicator of densely colonised host trees. More importantly, it is currently unclear how well verbenone emission is actually related to colonisation density and thus intraspecific competition. Here, we inoculated Norway spruce logs with I. typographus at two defined colonisation densities in the greenhouse and measured the emission of verbenone and its precursors α-pinene and verbenol over time. Verbenone emission was 3–7 times greater from colonised logs compared to decaying logs without beetles during the major part of larval development. Furthermore, our data supports the quantitative hypothesis, that the termination of attack on a tree is mediated by a cessation of the release of verbenol and continuous emission of verbenone. The latter is most likely a passively produced host-marking cue reflecting the actual density of conspecifics since per-beetle emission was unaffected by colonisation density. These findings shed new light on the regulation of bark beetle mass aggregations, which are currently causing previously unseen economic damages in temperate forests.

Galling insect communities mediate the effects of fire on their associated parasitoid communities

Abstract Understanding fire effects on multitrophic levels is critical to the context of changes in fire regime and climate. Insects located at higher trophic levels, like parasitoids, are more vulnerable to habitat changes than insect herbivores because they need well‐established populations of their hosts to survive. Currently, fire effects on parasitoids and their interactions with their hosts are unknown. Our aim was to study the changes in abundance, richness and species composition, as well as the food web structure in a system involving parasitoids and galling insects under different fire scenarios. We asked whether potential changes in abundance, species richness and composition of parasitoid communities are explained by galled plant abundance, species richness and composition of galling insects, and how fire affects the structure of galling insect–parasitoid food webs. The highest parasitoid richness was found in the 9 years after fire scenario, whereas parasitoid abundance was not affected by fire. The parasitoid species composition in the 9 years after fire scenario was different from that in the unburned and 3 years after fire scenarios. Parasitoid communities were modulated by galled plant abundance, species richness and composition of galling insect communities. Vulnerability was significantly higher in 9 years after fire scenarios, but it increased with increasing network size. Fire affects the community of parasitoids through changes in their host communities. Differences in exclusivity to fire scenarios and diet specialisation of parasitoid species and galling insect richness may explain the patterns found. Our study supports evidence that fire creates habitats heterogeneous in the availability of hosts shaping the parasitoid communities, with a bottom‐up effect in food webs. Additionally, in burned areas were recorded unique galling insect–parasitoid interactions.

The relationship between host plant traits and biodiversity across three sympatric seed‐feeding tri‐trophic systems in a tropical region of Brazil

Abstract We describe the diverse community of insect herbivores and parasitoids associated with the seeds and fruits of three sympatric Fabaceae species in Lavras, Minas Gerais, Brazil: Inga vera, Senna multijuga and Leucaena leucocephala. A total of 5353 individual insects, representing 77 different species, were identified via morphology and DNA barcoding. The non‐native L. leucocephala had the least diverse natural enemy community (N = 17 species), while two native species, I. vera and S. multijuga, were more diverse (N = 40 and N = 24 species, respectively). Individuals from the insect order Hymenoptera, a group dominated by parasitoid wasps, were the most diverse recovered from our samples in all hosts. Additionally, individual herbivore and parasitoid species were more likely to be found on a single host, with only four out of the 77 species found in more than one plant species. This includes the generalist agricultural pest herbivore, Lasioderma serricorne, found in two species, and three parasitoids connecting native S. multijuga and non‐native L. leucocephala communities, hinting at recent host shifts. Furthermore, different host plant traits had complex effects on the herbivore and parasitoid communities, where seed number per fruit promoted a propagating effect on the abundance and richness at both higher trophic levels, whereas seed and fruit weight did not. We highlight the importance of including parasitoids in insect community studies because they are highly diverse and provide important ecosystem services. Additional study of the biology, behaviour and distributions of parasitoids would be advantageous to inform conservation and biological control.

Tuta absoluta-Specific DNA in Domestic and Synanthropic Vertebrate Insectivore Feces.

The ecology of greenhouse pests generally involves parasitoid or predatory insects. However, we investigated whether the leaf miner Tuta absoluta (Meyrick, 1917) (Lepidoptera: Gelechiidae) is part of the diet of domestic and synanthropic vertebrate animals, such as birds, reptiles, and mammals, and that take part in an ecosystem that contains a high density of tomato greenhouses. Feces from domesticated partridges, common quails, and chickens, as well as from wild lizards were collected within tomato greenhouses, and fecal pellets from bats, swallows, common swifts, and house martins living in the vicinity of tomato greenhouses were collected outside. The efficiencies of three different DNA extraction methods were compared on bird, reptile, and mammal stool samples, and the DNA extracts were analyzed using probe real-time PCR for the presence of T. absoluta DNA. The results showed that bats fed on the pest, which was also part of the diet of several bird species: partridges and common quails kept within tomato greenhouses and swallows and common swifts living outside but in the vicinity of tomato greenhouses. In addition, fecal samples of three lizard species living near tomato crops also tested positive for T. absoluta DNA. The results suggest that aerial foraging bats and insectivorous birds are part of ecosystems that involve leaf miners and tomato greenhouses.

Diversity and ecology of the Family Orussidae (Insecta: Hymenoptera)

The biology of the Siricoidea superfamily, a sister group of the Symphyta and the family Orussidae (Parasitica: Woodwasps), can be imagined as the evolution of parasitoids would have been. The larvae of the Siricoidea wasps generally feed on dead wood that has been previously digested by a fungal symbiont. For this reason, many species carry spores of the fungus that they leave on the wood during oviposition. Diversity and ecology of parasitoid insects of the Family Orussidae (Insecta: Hymenoptera). In summary, basically, the following steps of analysis were covered: Exhaustive reading of each national and international article aiming at a global understanding and discovery of the approach used by its authors; Identification of the central ideas of each article; Classification of ideas around nuclei of meaning; Comparison between the different nuclei of meaning present in the studied articles; Classification of the nuclei of meaning in broader axes (themes) around which the authors' discussions revolved and (e) writing of the interpretative syntheses of the theme. Book scientific chapters, theses banks, university dissertations, national and international scientific articles, scientific journals book scientific chapters, theses banks, university dissertations, scientific journals and https://www.researchgate.net/post/How_to_increase_the_research_results_v... were also used. https://goo.gl/gLTTTs), HAL (https://hal.archives-ouvertes.fr/submit/index, SSRN (https://hq.ssrn.com/login/pubsigninjoin.cfm and ResearchGate (https ://www.researchgate.net/signup.SignUp.html).

Pest science in Pasteur’s Quadrant

AbstractMore than 25 years ago, Donald Stokes argued that we must move beyond the false dichotomy of basic or applied research and suggested that when considering a program of scientific research it is important to ask whether (i) the work is motivated by use and (ii) if there is a search for fundamental understanding. Giving yes/no answers to these questions allows us to characterize research more fully, replacing the “or” of “basic or applied” by a richer understanding of the process of science. Stokes proposed that research that was motivated by a consideration of use and sought fundamental understanding be called research in Pasteur’s Quadrant. One advantage of such work is that the search for fundamental understanding means that the problem-solving tools are more likely to be transferrable. After reviewing Stokes’s formulation of research, I illustrate it with examples from the control of tephritid flies and the use of insect parasitoids for biological control. Thinking about one’s work within Stokes’s framework has many advantages for individual scientists, including guidance for journal selection, how to organize and conclude papers and seminars, and the “elevator speech.” Furthermore, since research in Pasteur’s Quadrant has the characteristic of simultaneously increasing our understanding of how the world works and improving applications, it will more likely benefit the community of pest scientists.

Restricted diet prevents the induction of diapause in Trichogramma larvae

Photoperiodic and thermal diapause-inducing responses of insect parasitoids are well known whereas the trophic response is rather poorly studied. Our experiments showed that restricted diet prevented the induction of diapause in Trichogramma telengai parasitizing the grain moth (Sitotroga cerealella) eggs. In particular, the incidence of diapause decreased (1) with the rate of superparasitism (parasitoid:host ratios of 1:50, 1:25, 1:10, and 1:2 were tested), (2) with the time of host storage at 5 °C (0, 20, 40, 60, and 80 days), and (3) with host embryo development (1, 3, 5, 7, and 9 days at 20 °C). Both changes in the initial proportions of diapause-destined and non-diapause-destined individuals and differential mortality in these two categories were observed. These effects are most probably based on high nutritional demands of diapause-destined individuals: restricted diet causes nutrients deficiency and thereby prevents diapause. For the practice we conclude that if the induction of diapause for cold storage of mass or laboratory reared Trichogramma species is planned, host quality requirements should be tightened.

Potential of Hedgerows with Aromatic Plants as Reservoirs of Natural Enemies of Pests in Orange Orchards

In the present study, the potential of hedgerows from Mediterranean aromatic plant species, i.e., oregano, rosemary, sage, and savory, in orange field margins to function as reservoirs of natural enemies of citrus pests was tested in comparison to the common management practice of bare soil or weed vegetation. Assessments were based on the abundance and diversity of parasitoid wasps, spiders, and insect predators in the field margins and on the orange trees for two growing seasons. Savory plants harbored more parasitoids compared to weed vegetation and the other aromatic plants (savory > organic rosemary > sage > oregano). Weed vegetation hosted more arachnid predators than the aromatic plants in their first year in the orchard, but this was reversed with their full growth in the following year (most abundant on rosemary). Oregano and sage favor insect predators. The similarity of the natural enemy communities on the field margins and on the orange trees increased with time, indicating the insects' movement from the field margins to the trees. The results support the use of the tested aromatic plant species in conservation practices for targeted groups of beneficial arthropods in orange orchards, also considering the exploitation of suitable wild flowering plants of the weed flora.

Insect Pest Incidence with the System of Rice Intensification: Results of a Multi-Location Study and a Meta-Analysis

The System of Rice Intensification (SRI) developed in Madagascar has spread to many parts of the world, including India. This study assessing insect pest prevalence on rice grown with SRI vs. conventional methods at multiple locations in India was prompted by reports that SRI-managed rice plants are healthier and more resistant to pest and disease damage. Field experiments were conducted under the All-India Coordinated Rice Improvement Project over a 5-year period. The split-plot design assessed both cultivation methods and different cultivars, hybrids and improved varieties. Across the eight locations, SRI methods of cultivation showed a lower incidence of stem borer, planthoppers, and gall midge compared to conventional methods. Whorl maggots and thrips, on the other hand, were observed to be higher. Grain yield was significantly higher with SRI management across all locations. Higher ash, cellulose, hemicellulose, as well as silica content in rice plants under SRI management could explain at least in part the SRI plants’ resistance to pest damage. Analysis of guild composition revealed that in SRI plots, there were more natural enemies (insect predators and parasitoids) present and fewer crop pests (phytophages). A meta-analysis that considered other published research on this subject revealed a lower incidence of dead hearts, white ear-heads, and leaf folders, along with higher grain yield, in SRI plots.

Biodiversity of hymenopteran parasitoids

Parasitoid wasps are the most successful group of insect parasitoids, comprising more than half the known diversity of Hymenoptera and probably most of the unknown diversity. This lifestyle has enabled them to be used as pest control agents conferring substantial economic benefits to global agriculture. Major lineages of parasitoid wasps include Ichneumonoidea, Ceraphronoidea, Proctotrupomorpha, and a number of aculeate families. The parasitoid lifestyle arose only once among basal Hymenoptera, in the common ancestor of the Orussidae and Apocrita some 200+ Ma ago. The ancestral parasitoid wasp was probably an idiobiont on wood-living beetle larvae. From this comparatively simple biology, Hymenoptera radiated into an incredible diversity of hosts and parasitoid lifestyles, including hyperparasitoidism, kleptoparasitoidism, egg parasitoidism, and polyembryony, in several instances co-opting viruses to subdue their hosts. Many lineages evolved beyond the parasitoid niche, becoming secondarily herbivorous or predatory nest provisioners and eventually giving rise to most instances of insect societies.

A systematic review of the Families Chrysolampidae, Cleonymidae, Diparidae, Cerocephalidae, Chalcedectidae, Ceidae and Herbertiidae

Despite their small size, the Chalcidoidea play a significant role in the ecosystem. Most species are parasitoids of other insects, attacking the egg or larval stage of their host, although many other life cycles are known. These hosts can be found in at least 12 different insect orders, including Lepidoptera (butterflies and moths), Diptera (true flies), Coleoptera (beetles), Hemiptera (true insects) and other Hymenoptera, as well as two orders of Arachnida and even a family of Nematoda. In addition to the parasitoids, there are some species with the phytophagous habit, the larvae feed inside seeds, stems and galls. This work aims to examine the parasitoids of Chrysolampidae, Cleonymidae, Diparidae, Cerocephalidae, Chalcedectidae, Ceidae and Herbertiidae (Hymenoptera: Chalcidoidea). The present work used the reference of bibliographical research, understood as the act of inquiring and seeking information on a given subject, through a survey carried out in national and foreign databases, with the objective of detecting what there is of consensus or controversy. in the state of the art. Digital platforms were examined. Google Scholar, CrossRef, CiteFactor, Scilit (Scientific Literature), Academic Resource Index (Research), Scientific Indexing Services, ResearchGate, World Cat, DRJI (Directory of Research Journals Indexing), Semantic Scholar. The articles were published from 1972 to 2022 in indexed scientific research, book scientific chapters, theses banks, university dissertations, national and international scientific articles and scientific journals.

Competitive interactions in insect parasitoids: effects of microbial symbionts across tritrophic levels.

Competition for hosts is a common ecological interaction in insect parasitoids. In the recent years, it has become increasingly evident that microorganisms can act as 'hidden players' in parasitoid ecology. In this review, we propose that parasitoid competition should take into consideration the microbial influence. In particular, we take a tritrophic perspective and discuss how parasitoid competition can be modulated by microorganisms associated with the parasitoids, their herbivore hosts, or the plants attacked by the herbivores. Although research is still in its infancy, recent studies have shown that microbial symbionts can modulate the contest outcome. The emerging pattern is that microorganisms not only affect the competitive traits of parasitoids but also the fighting arena (i.e. the herbivore host and its food plant), in which competition takes place. We have also identified important gaps in the literature that should be addressed in future studies to advance our understanding about parasitoid competition.

Finding an egg in a haystack: variation in chemical cue use by egg parasitoids of herbivorous insects.

Egg parasitoids of herbivorous insects use an interplay of short- and long-range chemical cues emitted by hosts and host plants to find eggs to parasitize. Volatile compounds that attract egg parasitoids can be identified via behavioral assays and used to manipulate parasitoid behavior in the field for biological control of herbivorous pests. However, how and when a particular cue will be used varies over the life of an individual, as well as at and below species level. Future research should expand taxonomic coverage to explore variation in chemical cue use in more natural, dynamic settings. More nuanced understanding of the variability of egg parasitoid host-finding strategies will aid in disentangling the underlying genetics and further enhancing biological control.

Transcriptional regulation of host insulin signaling pathway genes controlling larval development by Microplitis manilae parasitization.

Idiobiont parasitoids using other insects as hosts sabotage the host growth and development to ensure their offspring survival. Numerous studies have discovered that insect development is subtly regulated by the conserved insulin signaling pathway. However, little is known about how wasp parasitization disrupts host development controlled by the insulin signaling pathway. Here we address this study to determine the effect of wasp parasitism on host Spodoptera frugiperda development using the idiobiont parasitoid Microplitis manilae as a model. Upon M. manilae parasitization, the body weight, body length, and food consumption of host insect were dramatically reduced compared to the unparasitized S. frugiperda. We next identified the core genes involved in host insulin signaling pathway and further analyzed the domain organizations of these genes. Phylogenetic reconstruction based on the insulin receptors clustered S. frugiperda together with other noctuidae insects. In the latter study, we profiled the expression patterns of host insulin signaling pathway genes in response to M. manilae parasitization at 2, 24,and 48 h, significant decreases in mRNA levels were recorded in S. frugiperda larvae upon 24and 48 h parasitization. These current findings substantially add to our understanding of the physiological interaction between parasitoid and host insects, thus contributing to revealing the molecular mechanism of parasitic wasps regulating host development.

Weeds Enhance Insect Diversity and Abundance and May Improve Soil Conditions in Mango Cultivation of South Florida.

This study examined if weeds could serve as insectary plants to increase beneficial insect abundance and diversity in mango cultivation in southern Florida. Additionally, we examined how weed presence affects mango tree soil health. We found that weeds significantly increased pollinating and parasitoid insect abundance and diversity. Eight insect orders and eighteen families were significantly more abundant on mango trees with weeds growing beneath them than those where weeds were removed. There was no difference in predatory insects between treatments, and slightly more herbivorous insects on weedy mango trees. Pollinating insects visiting mango flowers in the weed treatment were significantly greater, as well as spiders on weedy mango trees. However, there were more lacewings (Neuroptera) observed on the mango trees without weeds, and leaf chlorophyll in the old and new mango leaves was significantly greater, in the weed-free treatment. Soil conditions, however, significantly improved in soil carbon and a greater pH reduction in the presence of weeds, though weeds affected neither soil nitrogen, phosphorous, nor chlorophyll in productive green leaves. These results show that a tolerable level of selective weed species' presence may benefit insect, plant, and soil biodiversity in farms. This is important in increasing production, sustainability, and biodiversity in agriculture, which otherwise may be deficient in non-crop life.

Zieleń miejska Gdyni jako środowisko występowania parazytoidów z podrodziny Pimplinae (Hymenoptera, Ichneumonidae)

Urban greenery is defined by the high density and species diversity of plants. As such, it is a habitat for various groups of insects, including pests and insectivores. This study aims to determine the species composition and abundance of parasitoids of the subfamily Pimplinae in urban green spaces. The study was conducted between 2009 and 2016 in Gdynia. Sweep-net sampling and collecting dead insects from the beach were used. A total of 442 specimens belonging to 45 species of Pimplinae were collected. They represented 66.18% of the species listed in this habitat and 32.61% of the species found in Poland. The dominant species included Tromatobia lineatoria (14.26%), Scambus inanis (10.41%), Zaglyptus varipes (7.92%), Tromatobia ovivora (7.70%), Acropimpla pictipes (6.57%), Scambus calobatus (5.43%) and Gregopimpla inquisitor (5.21%). Three trophic groups were distinguished among the parasitoids, including parasitoids of phytophagous insects, i.e. Arachnida and Aculeata. Parasitoids of phytophagous insects were the most abundant [34 species (75.55%)].

The Secondary Parasitoid Perilampus tristis Mayr, 1905 and its Effect on the Primary Parasitoids on the Codling Moth, Cydia pomonella L.

Basheer, A., G. Ibrahim, N. Diab, K. Al-Assas, R. Alsaied Omar, F. Al-Ghammaz, H. Nasrallah and Y. Idris. 2023. The Secondary Parasitoid Perilampus tristis Mayr, 1905 and its Effect on the Primary Parasitoids on the Codling Moth, Cydia pomonella L. Arab Journal of Plant Protection, 41(2): 146-151. https://doi.org/10.22268/AJPP-41.2.146151 The study was carried out in some apple orchards infested with the codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae) in Khan Arnabah (Quneitra governorate) and Al-Hariseh (Sweida governorate). The study aimed to study the secondary parasitoid Perilampus tristis Mayr, 1905 (Hymenoptera: Perilampidae) and to identify its hosts of the primary parasitoids that parasitize the codling moth in the two study areas. Two methods were used to identify the insect parasitoids on the codling moth, (i) collecting infested fruits, and (ii) cardboard traps. The study revealed the presence of many insect parasitoids that parasitize the apple fruit worm, but the secondary parasitoid P. tristis parasitizes as a secondary parasitoid on the pupae of two types of primary parasitoids, the egg-larval parasitoid Ascogaster quadridentata Wesmael (Hymenoptera: Braconidae) and the young larval parasitoid Pristomerus vulnerator, Pzeran1799) (Hymenoptera: Ichneuomonidae) in the two study areas. The numbers of secondary parasitoids P. tristis increased in the cardboard traps, whereas the number of primary parasitoids A. quadridentata and P. vulnerator decreased in the two study areas. The secondary parasitoid P. tristis reduced the effectiveness of the primary parasitoids A. quadridentata and Pristomerus vulnerator in the two study areas. Keywords: apples, Khan Arnabah, Al Harisa, primary parasitoid, secondary parasitoid

Seasonal occurrence of Liriomyza bryoniae (Kaltenbach) and their parasitoids on cucumber crop

The results of this study revealed a steady increase in population density of leaf miners Liriomyza bryoniae (Kaltenbach) from 1.33 larvae/leaf at the beginning of the season to 3.3, 6, and 7 larvae/leaf with the progression of the agricultural season until 10 November, a slight drop in population occurred at the end of the season. The results revealed that the leaf miner population showed a significant negative correlation (-0.74) with maximum temperature and a non-significant negative correlation with minimum temperature. The maximum percentage of factor role was 55% for maximum temperature and ( 18 %) for the minimum temperature. Two species of insect parasitoids (Diglyphus isaea and Opius sp ) Hymenoptera order were recorded on the leafminer Liriomyza bryoniae larvae and pupae. Diglyphus isaea was the most common species. It was active throughout the growing season and increased in number as the leafminer populations increased. The rate of % parasitism was 4.3 % on 7 Aug. increased to 7.8, 12.8, 20.3, and 38.2% on 1 Sept., 7 Sept., 7 Oct., and 21 Oct respectively, while the rate of % parasitism of Opius sp ranged from 2.6% to 5.9% from 14 Sept to 21 Oct.

Characterization and biochemical activities of novel functional antimicrobial peptide (AMP) from Trichogramma chilonis

Introduction and Aim: The antimicrobial peptides (AMPs) are generally found in invertebrates, mammals, birds, plants and insects. AMPs produced by insect parasitoids contribute to innate immunity to resist infection due to lack of adaptive immunity. T. chilonis is one of the most effective endoparasitoid wasps for controlling lepidopterous insects. Several attempts have been made to isolate, characterize and develop a commercially viable product of AMPs from various insect sources. The present study aimed to characterize AMP from T. chilonis for potential antimicrobial and anti-cancer properties. Methods: AMP was identified through T. chilonis transcriptome sequence and designed in silico and synthesized. Its purity was quantified using RP-HPLC, and the mass identified by mass spectrophotometry. LC/MS-MS was employed to predict the sequence and the BLAST program used to compare the sequence. AMP was tested for haemolytic activity and antimicrobial activity. Two pathogenic bacteria and fungal strains were used and IC50 values and MIC values were predicted against microbial strains. Results: Synthetic peptide was found to be 95% homogenous with molecular weight of 3.48 kD. The peptide was identified to be a novel antimicrobial peptide consisting of 33 amino acid residues, and has a low computed instability index of -0.1.55 with high hydrophobic ratio of 27.27%. The antimicrobial activity revealed that T. chilonis antimicrobial peptide (TC-AMP) strongly inhibits the growth of selected human bacterial and fungal pathogens. While the haemolytic assay showed that the peptide did not obliterate human RBC in vitro. TC-AMP also showed an efficient inhibition of angiogenesis by in vivo model as evident by inhibition of vascularization. Conclusions: AMP derived from the parasitoid has a potent antibiotic and anti-angiogenesis property. The peptide can be used as a potential antimicrobial and anticancer drug in near future with more detailed studies on its targeted applications.

Indigenous plant extracts and their role in pest management in tea ecosystem

India is the world's second largest producer of black tea. Insects, mites, nematodes, and plant pathogenic diseases all damage the tea plant which severely hampered tea production causing about 10-15% crop loss every year. It would be 100% in extreme circumstances. In India, many synthetic pesticides are commonly employed to control tea pests. The use of an excessive amount pesticides could lead to a number of issues, including the development of resistance, negative impacts on non-target species such as insect predators and parasitoids, disruption of the ecological balance, and the accumulation of pesticide residues on tea leaves. In the international market, there is an increasing demand for organic tea or tea that is pesticide residue free, which influences the export price. Plant extracts having insecticidal qualities contains secondary metabolites such as alkaloids, terpenoids, phenolics, and minor secondary compounds can be used as an alternative to synthetic insecticides and these are environmentally safe, selective, biodegradable, cost-effective, and renewable options for use in IPM programmes in this context. Biopesticides are natural plant products that can be grown at a minimal cost by planters and extracted using indigenous methods.