Validation of deep amplicon sequencing of Dicrocoelium in small ruminants from Northern regions of Pakistan.

Dicrocoelium lancet flukes cause significant production loss in ruminant livestock. Although co-infection with multiple Dicrocoelium species within a host is common, techniques for studying the composition of these complex parasite communities are lacking. The pathogenicity, epidemiology, and therapeutic susceptibility of different helminth species vary, and little is known about the interactions that take place between co-infecting species and their hosts. Here, we describe the first applicationof metabarcoding deep amplicon sequencing method to studythe Dicrocoelium species in sheep and goats. First, rDNA ITS-2 sequences of four Dicrocoelium species (Dicrocoelium dendriticum, Dicrocoelium hospes, Dicrocoelium orientalis, and Dicrocoelium chinensis) were extracted from the NCBI public database. Phylogenetic analysis revealed separate clades of Dicrocoelium species; hence, molecular differentiation between each species is possible in co-infections. Second, 202 flukes belonging to seventeen host populations (morphologically verified as belonging to the Dicrocoelium genus) were evaluated to determine the deep amplicon sequencing read threshold of an individual fluke for each of the four species. The accuracy of the method in proportional quantification of samples collected from single hosts was further assessed. Overall, 198 (98.01%) flukes were confirmed as D. dendriticum and 1.98% produced no reads. The comparison of genetic distances between rDNA ITS-2 revealed 86% to 98% identity between the Dicrocoelium species. Phylogenetic analysis demonstrated a distinct clustering of species, apart from D. orientalis and D. chinensis, which sit very close to each other in a single large clade whereas D. hospes and D. dendriticum are separated into their own clade. In conclusion each sample was identified as D. dendriticum based on the proportion of MiSeq reads and validated the presence of this group of parasites in the Gilgit Baltistan and Khyber Pakhtunkhwa provinces of Pakistan. The metabarcoding deep amplicon sequencing technology and bioinformatics pathway have several potential applications, including species interactions during co-infections, identifying the host and geographical distribution of Dicrocoelium in livestock, drug therapy response evaluation and understanding of the emergence and spread of drug resistance.

Evaluation of the immunoregulatory effect of Dicrocoelium dendriticum eggs on inflammatory and anti-inflammatory cytokines in EAE model.

Experimental autoimmune encephalomyelitis (EAE) is an appropriate model for the study of the immunologic and pathologic mechanisms in multiple sclerosis (MS). According to the hygiene hypothesis, helminths can improve immunoregulation and have therapeutic effects on immune-mediated diseases. In this study, we used Dicrocoelium dendriticum (Dicrocoeliidae, Platyhelminthes) eggs for the evaluation of their prophylactic and treatment effects on EAE disease. D. dendriticum eggs were extracted. Female C57BL/6 mice were immunized with the specific antigen MOG35-55 , and then the egg extracts were utilized for prophylaxis and/or treatment. Clinical symptoms and other relevant parameters were assessed daily. The mRNA expression of transforming growth factor-ß (TGF-ß), interleukin-10 (IL-10), IL-6, IL-23 and IL-17 were assessed with a real-time polymerase chain reaction technique. Furthermore, secretion of TGF-ß and IL-17 cytokines were determined by enzyme-linked immunosorbent assay. Data indicated that clinical symptoms in prophylaxis and treatment groups were decreased significantly in comparison with the untreated control group (p < .001). Our results showed a significant decrease in IL-17, as well as an increase in TGF-ß cytokine in the treatment group compared to the EAE control group (p < .01). Furthermore, in the prophylaxis and treatment groups, the mRNA expression of disease-associated cytokines decreased and the mRNA expression of the anti-inflammatory cytokines increased. In this study, the D. dendriticum egg ameliorates the clinical symptoms of the EAE model through the modulation of related cytokines of Th17 and Treg cells. Therefore, using this parasite egg could be a new treatment for MS.

Molecular analysis of internal transcribed spacer 2 of Dicrocoelium dendriticum isolated from cattle, sheep, and goat in Iran.

BACKGROUND: Dicrocoelium dendriticum is a broadly distributed zoonotic helminth, which is mainly reported from domesticated and wild ruminants. There is little data covering the molecular features of this trematode; therefore, current study aimed to molecularly analyze D. dendriticum in livestock. METHODS: Totally, 23 samples of D. dendriticum were collected from cattle, sheep, and goat from Ilam, Lorestan, and Khuzestan, three west and south-west provinces of Iran from February to August 2018. After genomic DNA extraction, the internal transcribed spacer (ITS) 2 fragment was amplified and sequenced in samples. To investigate genetic variations through the ITS 2 fragment of obtained D. dendriticum, phylogenetic tree and network analysis were employed. RESULTS: All 23 samples were successfully amplified and sequenced. Phylogenetic tree showed that our samples were clearly grouped in a clade together with reference sequences. There was no grouping based on either geographical regions or hosts. Network analysis confirmed the phylogenetic findings and showed the presence of nine distinct haplotypes, while our samples together most of sequences, which were previously submitted to the GenBank, were grouped in the Hap1. CONCLUSIONS: Our findings indicated that although ITS 2 fragment discriminate D. dendriticum, this fragment is not suitable to study intra-species genetic variations. Therefore, exploring and describing new genetic markers could be more appropriate to provide new data about the genetic distribution of this trematode.

Dicrocoelium spp. in cattle from Wa, Ghana: prevalence and phylogeny based on 28S rRNA.

Dicrocoeliosis is a trematode infection in cattle, sheep and goats caused by the small liver fluke, Dicrocoelium spp. Though endemic in Ghana, its disease situation is poorly understood. In the present study, the prevalence, distribution and worm load of Dicrocoelium spp. in cattle at slaughter in Wa were determined. A total of 389 cattle were screened during meat inspection for liver flukes, and polymerase chain reaction accompanied by DNA sequencing of the 28S rRNA gene was used to identify Dicrocoelium spp. Generally, prevalence of bovine dicrocoeliosis (small liver fluke) stood at 19.54 % with prevalence in males and females being 17.62 % and 21.43 %, respectively. Animals under 2 years suffered more infection than older ones (23.08 % vs. 16.80 %). Dicrocoelium infection was recorded in animals from all the communities where slaughtered cattle came from. On average, 31 flukes per infected animal were recorded. A molecular confirmatory test on seven flukes identified them as D. hospes. This preliminary study highlights the importance of bovine dicrocoeliosis in Ghana and has identified D. hospes as a causal agent. The data provides basis for further studies to appraise the trematode disease situation in animals and phylogeny of Dicrocoelium spp. circulating in Ghana.

Molecular confirmation of Dicrocoelium dendriticum in the Himalayan ranges of Pakistan.

Lancet liver flukes of the genus Dicrocoelium (Trematoda: Digenea) are recognised parasites of domestic and wild herbivores. The aim of the present study was to confirm the species identity of Dicrocoeliid flukes collected from the Chitral valley in the Himalayan ranges of Pakistan. The morphology of 48 flukes belonging to eight host populations was examined; but overlapping traits prevented accurate species designation. Phylogenetic comparison of published D. dendriticum ribosomal cistron DNA, and cytochrome oxidase-1 (COX-1) mitochondrial DNA sequences with those from D. chinensis was performed to assess within and between species variation and re-affirm the use of species-specific single nucleotide polymorphism markers. PCR and sequencing of 34 corresponding fragments of ribosomal DNA and 14 corresponding fragments of mitochondrial DNA from the Chitral valley flukes, revealed 10 and 4 unique haplotypes, respectively. These confirmed for the first time the molecular species identity of Pakistani lancet liver flukes as D. dendriticum. This work provides a preliminary illustration of a phylogenetic approach that could be developed to study the ecology, biological diversity, and epidemiology of Dicrocoeliid lancet flukes when they are identified in new settings.

Molecular characterization and immunodiagnostics of Dicrocoelium dendriticum species isolated from sheep of north-west Himalayan region.

Despite its extensive presence among grazing ruminants, dicrocoeliosis, also known as 'small liver fluke' disease, is poorly known and often underestimated by researchers and practitioners in many countries. The accurate identification and prepatent diagnosis of Dicrocoelium dendriticum infection is an essential prerequisite for its prevention and control. In the present study, the morphologically identified specimens isolated from the bile ducts of sheep (Ovis aries) were validated through molecular data. The sequence analysis of the second internal transcribed spacer (ITS-2) of our isolates showed a high degree of similarity with D. dendriticum using the BLAST function of the National Center for Biotechnology Information (NCBI). The phylogenetic analysis of our isolates showed a close relationship with previously described D. dendriticum isolates from different countries. The antigenic profiles of somatic and excretory/secretory (E/S) antigens of D. dendriticum were revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using sera from sheep naturally infected with D. dendriticum. By SDS-PAGE, 16 distinct bands were revealed from crude somatic fraction. Immunoblotting analysis of these proteins with positive sera exhibited six seroreactive bands ranging from 27 to 130 kDa. Among these, the 84 and 130 kDa bands were quite specific, with high diagnostic specificity and sensitivity. The E/S fraction comprised nine distinct bands, as revealed by SDS-PAGE analysis. Immunoblotting analysis of these proteins with positive sera exhibited five antigenic bands ranging from 27 to 130 kDa. Among these, the 130 kDa band was found to be quite specific, with high diagnostic specificity and sensitivity. The present study concludes that the protein bands of 84 and 130 kDa in somatic fraction and 130 kDa in E/S fraction can be used for the immunodiagnostic purpose for this economically important parasite, which may also encourage further studies regarding their vaccine potential.

Molecular Characterization of Fasciola and Dicrocoelium Species Isolated from Ruminant Livestock in Qazvin, Iran.

INTRODUCTION: Fascioliasis and dicrocoeliasis are the most frequent zoonotic diseases with increasing human health problems in different parts of Iran. Two species, Fasciola hepatica (F. hepatica) and Fasciola gigantica (F. gigantica), are spread in the country. Molecular approaches have a decisive role in identifying both the species. The aim of this study was to detect Fasciola spp. and Dicrocoelium spp. by amplifying the ITS-2 and 28S rDNA gene sequence. METHODS: Overall, 30 infected liver samples were collected from the livestock of Qazvin, Iran. The adult flukes were collected from different livestock. DNA extraction and PCR amplification of ribosomal RNA gene region (ITS2) and 28S rDNA gene fragment were conducted and a phylogenetic tree was constructed. RESULT: All the isolates obtained from the cattle (No: 7) and 82.6% (No: 19) of sheep isolates were infected with F. hepatica species, whereas 17.4% (No: 4) of sheep isolates were infected with F. gigantica. It was also shown that F. hepatica was the predominant species of Fasciola present in the region. All the specimens were infected with Dicrocoelium dendriticum (D. dendriticum). CONCLUSION: Both the species of Fasciola were found in Qazvin. D. dendriticum was the sole infecting species of the Dicrocoelium genus in the livestock of the city of Qazvin. Further research studies are needed to determine the intermediate host of the parasites in the region.

Dicrocoeliosis in extensive sheep farms: a survey.

BACKGROUND: This study investigated the epidemiological and molecular aspects of dicrocoeliosis in extensive sheep farms. METHODS: From 2013 to 2014, copromicroscopical analyses in 190 dairy sheep farms and anatomo-pathological inspections in six slaughterhouses were carried in Sardinia, Italy. Rectal faecal samples were analyzed using the FLOTAC® method, and anatomo-pathological examinations were based on detecting thickened terminal bile ducts (TTBDs). In addition, genetic analyses were conducted on representative DNA samples of adult Dicrocoelium spp. RESULTS: Ninety-seven (51.1%) out of 190 sheep farms were coprologically positive for Dicrocoelium spp. In the liver, on the surface and cut surface, TTBDs were reported in 40.1% (309/770) and 15.3% (118/770) of the animals examined, respectively, with an overall prevalence of 25.5% (196/770). No intraspecific genetic variation was observed among the Dicrocoelium dendriticum isolates. CONCLUSIONS: Our survey reveals the widespread presence of D. dendriticum in Sardinia, although seasonal, geographical and climatic conditions might be key factors in modulating the infection prevalence. Examining typical lesions due to D. dendriticum in the liver in abattoirs can be used as a marker for tracking chronic dicrocoeliosis infection.

Phylogenetic relationships between Dicrocoelium chinensis populations in Japan and China based on mitochondrial nad1 gene sequences.

We carried out phylogenetic analyses of the relationships between Dicrocoelium chinensis populations in Japan and China using molecular markers. One hundred nine lancet flukes collected from Japan and China were identified as D. chinensis based on their testis orientation and the nucleotide sequences of their ribosomal ITS2. These flukes were analyzed phylogenetically using mitochondrial nad1 gene sequences. An analysis of molecular variance found that the percentage of variation between the countries was extremely high, indicating that the D. chinensis populations in Japan and China are differentiated genetically. D. chinensis mainly parasitizes wild sika deer, which is thought to originate in northeast Asia and to have colonized into Japan from the Eurasia continent in the Pleistocene glaciations. In addition, phylogenic analyses indicated that Japanese sika deer is genetically differentiated from Chinese population; therefore, we hypothesize that D. chinensis might have been introduced into Japan along with the migration of infected wild ruminants in the Pleistocene, and then the population became differentiated from the Chinese population. This study provides the nucleotide sequences of the nad1 gene of D. chinensis in Japan for the first time and shows that these sequences are useful for elucidating the phylogenetic relationships of the Dicrocoelium species prevalent in Asia.

Population genetic analysis informs the invasion history of the emerging trematode Dicrocoelium dendriticum into Canada.

Parasite distributions are constantly changing due to climate change, local and global movement of animals and humans, as well as land use and habitat change. The trematode Dicrocoelium dendriticum is a relatively recent invader of Canada, being first reported in eastern Canada in the 1930s and western Canada in the 1970s. However, historical records are scarce and its emergence is poorly understood. The establishment of this parasite in Canada provides an interesting opportunity to explore the use of population genetic approaches to help elucidate the invasion history of a relatively recently established helminth parasite. In this study, we compare the genetic diversity and population structure of a number of D. dendriticum populations from western and eastern Canada, and compare these with much longer established European populations. Two independent genetic marker systems were used; a microsatellite marker panel and a cytochrome c oxidase 1 (cox1) mitochondrial (mt)DNA sequence marker. We found distinct differences in both genetic diversity and population structure of the different Canadian populations that provide insights into their invasion histories compared with the European populations. Two populations from British Columbia, Canada - Salt Spring and Vancouver Islands - are of low diversity, show evidence of a population bottleneck and are closely related to each other, suggesting a shared recent history of establishment. These west coast populations are otherwise most closely related to those from eastern Canada and western Europe, and in contrast are genetically divergent from those in Cypress Hills, Alberta, Canada. Although the Alberta parasite population is the most recently reported in Canada, being first identified there in the early 1990s, it was the most genetically diverse of those examined and showed a strong pattern of admixture of genotypes present in western and eastern Europe. Overall, our results are consistent with a model in which western Europe is likely the source of flukes on the east coast of Canada, which were then subsequently translocated to the west coast of Canada. The most recently reported D. dendriticum population in Canada appears to have a different history and likely has multiple origins.

Evaluation of molecular methods for the field study of the natural history of Dicrocoelium dendriticum.

There is a need for improved methods for the study of the impacts of climatic and livestock management change on the epidemiology of production-limiting helminth parasitic diseases. In this study we report the application of molecular methods to describe the natural history of the small lancet fluke, Dicrocoelium dendriticum on Machair pastures on the Inner Hebridean Isle of Coll. Our results build upon those of the only previous historic field study of D. dendriticum in the British Isles that had been undertaken on the same study site. We demonstrate the value of combining conventional parasitological methods with PCR amplification of a mitochondrial DNA fragment for the detection of D. dendriticum in ants and snails, and PCR amplification of ITS2 and 28S ribosomal DNA fragments to support the species identity of the intermediate hosts, to improving understanding of the epidemiology of D. dendriticum. We report the presence of D. dendriticum infection in cattle, sheep and rabbits grazing on Machair pastures. D. dendriticum infection was identified in a high percentage of the snails, identified as Cochlicella acuta and Cernuella virgata, and in a high percentage of Formica fusca and Myrmica ruginoides ants that were collected from, or clinging to, the tops of flowers. We have identified the involvement of different intermediate host species and higher prevalences of snail and ant infection than previously reported, in part reflecting differences between the sensitivity and specificity of morphological and molecular speciation methods. Overall, our results highlight the complex life history of dicrocoeliosis and illustrate the parasite's generalist host strategy that confers potential to exploit new niches created by climatic change or grazing management for habitat conservation.

On the presence and immunoregulatory functions of extracellular microRNAs in the trematode Fasciola hepatica.

Liver flukes represent a paraphyletic group of endoparasitic flatworms that significantly affect man either indirectly due to economic damage on livestock or directly as pathogens. A range of studies have focussed on how these macroscopic organisms can evade the immune system and live inside a hostile environment such as the mammalian liver and bile ducts. Recently, microRNAs, a class of short noncoding gene regulators, have been proposed as likely candidates to play roles in this scenario. MicroRNAs (miRNAs) are key players in development and pathogenicity and are highly conserved between metazoans: identical miRNAs can be found in flatworms and mammalians. Interestingly, miRNAs are enriched in extracellular vesicles (EVs) which are secreted by most cells. EVs constitute an important mode of parasite/host interaction, and recent data illustrate that miRNAs play a vital part. We have demonstrated the presence of miRNAs in the EVs of the trematode species Dicrocoelium dendriticum and Fasciola hepatica (Fhe) and identified potential immune-regulatory miRNAs with targets in the host. After our initial identification of miRNAs expressed by F. hepatica, an assembled genome and additional miRNA data became available. This has enabled us to update the known complement of miRNAs in EVs and speculate on potential immune-regulatory functions that we review here.

Characterization of nine microsatellite loci for Dicrocoelium dendriticum, an emerging liver fluke of ungulates in North America, and their use to detect clonemates and random mating.

This study characterizes polymorphic microsatellite loci from adults of the liver fluke Dicrocoelium dendriticum sampled from a population of sympatric beef cattle and wapiti in a region of emergence in southern Alberta, Canada. We also scrutinized the markers to validate their use in studying the population genetics of this complex life cycle parasite. Among the nine loci described, four deviated significantly from Hardy Weinberg Equilibrium (HWE) due to technical artefacts. The remaining five loci were in HWE. These five provided sufficient resolution to identify clonemates produced from the obligate asexual reproduction phase of the life cycle in snails and to assess the impact of non-random transmission of clonemates on measures of FIS, FST and genotypic disequilibrium. Excluding clonemates, we show that the sub-population of worms was in HWE, that average FIS within hosts was 0.003 (p=0.4922) and that there was no population genetic structure among hosts FST=0.001 (p=0.3243). These markers will be useful for studies of Dicrocoelium dendriticum ecology, transmission, and evolution.

Characterization of Dicrocoelium dendriticum haplotypes from sheep and cattle in Iran based on the internal transcribed spacer 2 (ITS-2) and NADH dehydrogenase gene (nad1).

The present study assessed whether the genetic variation among different hosts (sheep and cattle) and geographical isolates (n= 28) of Dicrocoelium dendriticum from Iran is present based on mitochondrial (nad1) and ribosomal (ITS-2) DNA markers. Molecular analysis revealed the presence of at least ten and two distinct haplotypes in the NADH dehydrogenase gene (nad1) and internal transcribed spacer 2 (ITS-2), respectively. The nad1 and ITS-2 sequence data were deposited in GenBank under accession numbers, JX050110-134 and JQ966972-3. According to the results of our study, ND-D and ITS-A are established as being the predominant haplotypes of D. dendriticum in Iran. The Iranian isolates showed a higher intraspecific genetic diversity of 0-0.97% for nad1, compared to 0-0.42% for ITS-2. The alignment and comparison of nad1 and ITS-2 sequences revealed eight and one polymorphic sites, respectively. In the nad1 sequences, six were silent and two nucleotide substitutions were responsible for amino acid alterations. A phylogenetic analysis of the sequence data revealed that host associations and geographic location are not likely useful markers for D. dendriticum haplotype classification. Consequently, sequencing results obtained from the nad1 gene as a mitochondrial marker for the first time in this study would provide a valuable tool to analyse further molecular details of D. dendriticum worldwide.

Characterization of Dicrocoelium dendriticum isolates from small ruminants in Shaanxi Province, north-western China, using internal transcribed spacers of nuclear ribosomal DNA.

The genetic variations in internal transcribed spacers (ITS) spanning ITS-1, 5.8S and ITS-2 rDNA of Dicrocoelium dendriticum, isolated from sheep and goats in four geographical regions in Shaanxi province, were examined. The lengths of ITS-1, 5.8S and ITS-2 rDNA sequences for D. dendriticum were 749 bp, 161 bp and 234 bp, respectively. Intra-specific sequence variations of D. dendriticum were 0-0.5% for ITS-1 and 0-1.3% for ITS-2 rDNA, while the inter-specific variations among species in genus Dicrocoelium in ITS-2 rDNA were 3.4-12.3%. Phylogenetic analysis based on sequences of ITS-2 rDNA showed that all D. dendriticum isolates in the present study were grouped with reference D. dendriticum isolates from sheep and goats, and D. dendriticum isolates from cattle and Japanese serow were clustered in a sister clade. However, the phylogenetic tree could not reveal geographically genetic relationships of D. dendriticum isolates in different origins and hosts. These findings provided basic information for further study of molecular epidemiology and control of D. dendriticum infection in Shaanxi province as well as in the world.

Distinct distribution of Dicrocoelium dendriticum and D. chinensis in Iwate Prefecture, Japan, and a new final host record for D. chinensis.

This study dealt with the morphological and molecular identification of Dicrocoelium flukes obtained from Japanese serow (Capricornis crispus) and sika deer (Cervus nippon centralis) in the twelve districts of Iwate Prefecture, Japan. Dicrocoelium dendriticum and D. chinensis were exclusively detected in the western, and coastal and eastern areas of Iwate Prefecture, respectively. This geographically distinct occurrence of the two Dicrocoelium species would be associated with the distribution of the final hosts, sika deer for D. chinensis and Japanese serow for D. dendriticum. This study also reports that Capricornis crispus is a new final host of D. chinensis.

Dicrocoelium chinensis and Dicrocoelium dendriticum (Trematoda: Digenea) are distinct lancet fluke species based on mitochondrial and nuclear ribosomal DNA sequences.

Lancet flukes parasitize the bile ducts and gall bladder of a range of mammals, including humans, causing dicrocoeliosis. In the present study, we sequenced and characterized the complete mitochondrial (mt) genomes as well as the first and second internal transcribed spacers (ITS-1 and ITS-2=ITS) of nuclear ribosomal DNA (rDNA) of two lancet flukes, Dicrocoelium chinensis and D. dendriticum. Sequence comparison of a conserved mt gene and nuclear rDNA sequences among multiple individual lancet flukes revealed substantial nucleotide differences between the species but limited sequence variation within each of them. Phylogenetic analysis of the concatenated amino acid and multiple mt rrnS sequences using Bayesian inference supported the separation of D. chinensis and D. dendriticum into two distinct species-specific clades. Results of the present study support the proposal that D. dendriticum and D. chinensis represent two distinct lancet flukes. While providing the first mt genomes from members of the superfamily Plagiorchioidea, the novel mt markers described herein will be useful for further studies of the diagnosis, epidemiology and systematics of the lancet flukes and other trematodes of human and animal health significance.

Surface analysis of Dicrocoelium dendriticum. The molecular characterization of exosomes reveals the presence of miRNAs.

With the aim of characterizing the molecules involved in the interaction of Dicrocoelium dendriticum adults and the host, we have performed proteomic analyses of the external surface of the parasite using the currently available datasets including the transcriptome of the related species Echinostoma caproni. We have identified 182 parasite proteins on the outermost surface of D. dendriticum. The presence of exosome-like vesicles in the ESP of D. dendriticum and their components has also been characterized. Using proteomic approaches, we have characterized 84 proteins in these vesicles. Interestingly, we have detected miRNA in D. dendriticum exosomes, thus representing the first report of miRNA in helminth exosomes. BIOLOGICAL SIGNIFICANCE: In order to identify potential targets for intervention against parasitic helminths, we have analyzed the surface of the parasitic helminth Dicrocoelium dendriticum. Along with the proteomic analyses of the outermost layer of the parasite, our work describes the molecular characterization of the exosomes of D. dendriticum. Our proteomic data confirm the improvement of protein identification from "non-model organisms" like helminths, when using different search engines against a combination of available databases. In addition, this work represents the first report of miRNAs in parasitic helminth exosomes. These vesicles can pack specific proteins and RNAs providing stability and resistance to RNAse digestion in body fluids, and provide a way to regulate host-parasite interplay. The present data should provide a solid foundation for the development of novel methods to control this non-model organism and related parasites. This article is part of a Special Issue entitled: Proteomics of non-model organisms.