Non-specific transient mutualism between the plant parasitic nematode, Bursaphelenchus xylophilus , and the opportunistic bacterium Serratia quinivorans BXF1, a plant-growth promoting pine endophyte with antagonistic effects

The aim of this study is to understand the biological role of Serratia quinivorans BXF1, a bacterium commonly found associated with Bursaphelenchus xylophilus, the plant parasitic nematode responsible for pine wilt disease. Therefore, we studied strain BXF1 effect in pine wilt disease. We found that strain BXF1 promoted in vitro nematode reproduction. Moreover, the presence of bacteria led to the absence of nematode chitinase gene (Bxcht-1) expression, suggesting an effect for bacterial chitinase in nematode reproduction. Nevertheless, strain BXF1 was unable to colonize the nematode interior, bind to its cuticle with high affinity or protect the nematode from xenobiotic stress. Interestingly, strain BXF1 was able to promote tomato and pine plant-growth, as well as to colonize its interior, thus, acting like a plant-growth promoting endophyte. Consequently, strain BXF1 failed to induce wilting symptoms when inoculated in pine shoot artificial incisions. This bacterium also presented strong antagonistic activities against fungi and bacteria isolated from Pinus pinaster. Our results suggest that B. xylophilus does not possess a strict symbiotic community capable of inducing pine wilt disease symptoms as previously hypothesized. We show that bacteria like BXF1, which possess plant-growth promoting and antagonistic effects, may be opportunistically associated with B. xylophilus, possibly acquired from the bacterial endophytic community of the host pine.

he genome and genetics of a high oxidative stress tolerant Serratia sp. LCN16 isolated from the plant parasitic nematode Bursaphelenchus xylophilus

Background: Pine wilt disease (PWD) is a worldwide threat to pine forests, and is caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus. Bacteria are known to be associated with PWN and may have an important role in PWD. Serratia sp. LCN16 is a PWN-associated bacterium, highly resistant to oxidative stress in vitro, and which beneficially contributes to the PWN survival under these conditions. Oxidative stress is generated as a part of the basal defense mechanism used by plants to combat pathogenic invasion. Here, we studied the biology of Serratia sp. LCN16 through genome analyses, and further investigated, using reverse genetics, the role of two genes directly involved in the neutralization of H2O2, namely the H2O2 transcriptional factor oxyR; and the H2O2-targeting enzyme, catalase katA. Results: Serratia sp. LCN16 is phylogenetically most closely related to the phytosphere group of Serratia, which includes S. proteamaculans, S. grimessi and S. liquefaciens. Likewise, Serratia sp. LCN16 shares many features with endophytes (plant-associated bacteria), such as genes coding for plant polymer degrading enzymes, iron uptake/ transport, siderophore and phytohormone synthesis, aromatic compound degradation and detoxification enzymes. OxyR and KatA are directly involved in the high tolerance to H2O2 of Serratia sp. LCN16. Under oxidative stress, Serratia sp. LCN16 expresses katA independently of OxyR in contrast with katG which is under positive regulation of OxyR. Serratia sp. LCN16 mutants for oxyR (oxyR::int(614)) and katA (katA::int(808)) were sensitive to H2O2 in relation with wild-type, and both failed to protect the PWN from H2O2-stress exposure. Moreover, both mutants showed different phenotypes in terms of biofilm production and swimming/swarming behaviors. Conclusions: This study provides new insights into the biology of PWN-associated bacteria Serratia sp. LCN16 and its extreme resistance to oxidative stress conditions, encouraging further research on the potential role of this bacterium in interaction with PWN in planta environment.

Characterization of glutathione S transferases from the plant-parasitic nematode, Bursaphelenchus xylophilus

We have previously identified two secreted glutathione S-transferases (GST) expressed in the pharyngeal gland cell of Bursaphelenchus xylophilus, which are upregulated post infection of the host. This study examines the functional role of GSTs in B. xylophilus biology. We analysed the expression profiles of all predicted GSTs in the genome and the results showed that they belong to kappa and cytosolic subfamilies and the majority are upregulated post infection of the host. A small percentage is potentially secreted and none is downregulated post infection of the host. One secreted protein was confirmed as a functional GST and is within a cluster that showed the highest expression fold change in infection. This enzyme has a protective activity that may involve host defences, namely in the presence of terpenoid compounds and peroxide products. These results suggest that GSTs secreted into the host participate in the detoxification of host-derived defence compounds and enable successful parasitism.

Identification and characterization of parasitism genes from the pinewood nematode Bursaphelenchus xylophilus reveals a multilayered detoxification strategy.

The migratory endoparasitic nematode Bursaphelenchus xylophilus, which is the causal agent of pine wilt disease, has phytophagous and mycetophagous phases during its life cycle. This highly unusual feature distinguishes it from other plantparasitic nematodes and requires profound changes in biology between modes. During the phytophagous stage, the nematode migrates within pine trees, feeding on the contents of parenchymal cells. Like other plant pathogens, B. xylophilus secretes effectors from pharyngeal gland cells into the host during infection.We provide the first description of changes in the morphology of these gland cells between juvenile and adult life stages. Using a comparative transcriptomics approach and an effector identification pipeline, we identify numerous novel parasitism genes which may be important for the mediation of interactions of B. xylophilus with its host. In-depth characterization of all parasitism genes using in situ hybridization reveals two major categories of detoxification proteins, those specifically expressed in either the pharyngeal gland cells or the digestive system. These data suggest that B. xylophilus incorporates effectors in a multilayer detoxification strategy in order to protect itself from host defence responses during phytophagy.

POPULATION DYNAMICS OF PLANT PARASITIC NEMATODES AND THEIR RELATIONSHIPS WITH SOIL PHYSICO-CHEMICAL PARAMETERS DURING THE FALLOW PERIOD AFTER SUGARBEET AND LEADING INTO TOMATO CULTIVATION

The main objective of this research was the study of the soil nematode community, and in particular plant parasitic nematodes (PPN), from a field located in Portugal’s southern region, used for sugarbeet production. The study was performed from February to July 2003, covering part of the fallow period previous to tomato cultivation, the alternative crop in the rotation. The end of the fallow period in March and the soil preparation period in May were marked by a significant reduction in the numbers of PPN, whereas their numbers increased on the following tomato crop. The genus Helicotylenchus stood out as the most representative group, forming 90% of all PPN counted each month. The genus Heterodera was relatively abundant in the months following the previous sugarbeet crop, and numbers of the genus Meloidogyne increased during the tomato crop. The correlations between these group and environmental parameters show that, apart from the direct influence of the host, pH, organic matter, temperature and soil moisture significantly influenced nematode abundance and community composition.

Computerized key of the genus genus Laimaphelenchus Fuchs, 1937

The Order Aphelenchida contains several genera of economic importance, namely Aphelenchoides and Bursaphelenchus. Nematode species belonging to these 2 genera frequently co-habit with other genera such as Laimaphelenchus. It is therefore important to clearly distinguish them, as well as understand the group´s biodiversity. A computerized, or e-key, for the genus Laimaphelenchus Fuchs has been developed in the BiKey Identification system (Dianov & Lobanov, 1996-2004). The e-key includes 14 species and 34 characters (from 2 to 6 character states each). It also includes the built-in algorithm ranging characters according their diagnostic values to minimize the number of the diagnosis steps (average number of steps is 2.7; values are re-calculated at each step). The most important characters (as calculated by BiKey) are: length of posterior branch of the female genital system; excretory pore position; vulval anterior flap shape; number pairs of mail tale papillae; male bursa shape (ventral view); number of tail tip setae in female; female tail tip stub shape; presence of mucro on tail tip in male. Key is pictorial (image-operating), multientry, as other BiKey products.

Chromosome structure and behaviour in Bursaphelenchus xylophilus (Nematoda: Parasitaphelenchidae) germ cells and early embryo

Chromosome structure and behaviour in both meiosis of the germ cells and mitosis of the embryo from fertilisation to the two-cell stage in Bursaphelenchus xylophilus were examined by DAPI staining and three-dimensional reconstruction of serial-section images from confocal laser-scanning microscopy. By this method, each chromosome’s shape and behaviour were clearly visible in early embryogenesis from fertilisation through the formation and fusion of the male and female pronuclei to the first mitotic division. The male pronucleus was bigger than that of the female, although the oocyte is larger and richer in nutrients than the sperm. From the shape of the separating chromosomes at anaphase, the mitotic chromosomes appeared to be polycentric or holocentric rather than monocentric. Each chromosome was clearly distinguishable in the male and female germ cells, pronuclei of the one-cell stage embryo, and the early embryonic nuclei. The haploid number of chromosomes (N) was six (2n = 12), and all chromosomes appeared similar. The chromosome pair containing the ribosomal RNA-coding site was visualised by fluorescence in situ hybridisation. Unlike the sex determination system in Caenorhabditis elegans (XX in hermaphrodite and XO in male), the system for B. xylophilus may consist of an XX female and an XY male.

Bursaphelenchus xylophilus : opportunities in comparative genomics and molecular host–parasite interactions

Most Bursaphelenchus species are fungal feeding nematodes that colonize dead or dying trees. However, Bursaphelenchus xylophilus , the pine wood nematode, is also a pathogen of trees and is the causal agent of pine wilt disease. B. xylophilus is native to North America and here it causes little damage to trees. Where it is introduced to new regions it causes huge damage. The most severely affected areas are found in the Far East but more recently B. xylophilus has been introduced into Portugal and the potential for damage here is also high. As incidence and severity of pine wilt disease are linked to temperature we suggest that climate change is likely to exacerbate the problems caused by B. xylophilus and, in addition, will extend (northwards in Europe) the range in which pine wilt disease can occur. Here we review what is currently known about the interactions of B. xylophilus with its hosts, including recent developments in our understanding of the molecular biology of pathogenicity in the nematode. We also examine the potential developments that could be made by more widespread use of genomics tools to understand interactions between B. xylophilus , bacterial pathogens that have been implicated in disease and host trees.

Review of the genus Bursaphelenchus Fuchs, 1937 (Nematoda, Aphelenchida) with some conclusions on the host-parasite and vector-parasite evolution.

INTRODUCTION AND GOALS: Genus Bursaphelenchus includes several pests of the world importance for the rural economy, the most dangerous are the Bursaphelenchus xylophilus (the pinewood nematode caused decline of the pine trees in south Asia and in one spot area in Europe, Portugal, Peninsula de Setubal) and the Bursaphelenchus cocophilus, causing the decline of coco-palm plantations in Carribean and Latin American regions. The peculiarity of the host-parasite association of the genus that the nematode life cycle includes three trophic components: plant (mostly a tree), insect vector and a fungus. Goals of the presentation is to list all species of the world fauna and all efficient diagnostic characters, then create the identification tool and analyze the similarity of species and possible ways and causes of the host-parasite evolution of the group. RESULTS: Complete list of species with synonymy and a catalogue of all efficient diagnostic characters with their states, selected from papers of the most experienced taxonomists of the genus, are given for the genus Bursaphelenchus. List of known records of Bursaphelenchus species with names of natural vectors and plants and their families is given (for world pests the most important groups of trees and insects are listed). The tabular, traditional and computer-aided keys are presented. Dendrograms of species relationships (UPGMA, standard distance: mean character difference) based on all efficient taxonomic characters and separately on the spicule characters only, are given. Discussion whether the species groups are natural or purely diagnostic ones is based on the relationships dendrograms and the vector and associated plant ranges of Bursaphelenchus species; the xylophilus species group (B. xylophilus, B. abruptus, B. baujardi, B. conicaudatus, B. eroshenkii, B. fraudulentus, B. kolymensis, B. luxuriosae; B. mucronatus), the hunti group (B. hunti, B. seani, B. kevini and B. fungivorus) are probably the natural ones. CONCLUSIONS: The parasitic nematode association includes three trophic components: plant, insect vector and fungus. The initial insect-plant complex Scolytidae-Pinaceae is changeable and only in rare occasions the change of the preferred vector to Cerambycidae (the xylophilus group), Hymenoptera (the hunti group) led to formation of the natural species-groups. From the analysis it is clear that although the vector range is changeable it is comparatively more important for the evolution of the genus Bursaphelenchus than associations with plants at the family level. Data on the fungi species (3rd component in natural Bursaphelenchus associations) are insufficient for the detailed comparative analysis.

Pine wilt disease: a worldwide threat to forest ecosystems

The pinewood nematode (PWN), Bursaphelenchus xylophilus, the causal agent of pine wilt disease (PWD), is a serious pest and pathogen of forest tree species, in particular among the genus Pinus. It was first reported from Japan in the beginning of the XXth century, where it became the major ecological catastrophe of pine forests, with losses reaching over 2 million m3/ year in the 1980s. It has since then spread to other Asian countries such as China, Taiwan and Korea, causing serious losses and economic damage. In 1999, the PWN was first detected in the European Union (EU), in Portugal, and immmediately prompted several government (national and EU) actions to assess the extent of the nematode’s presence, and to contain B. xylophilus and its insect vector (Monochamus galloprovincialis) to an area with a 30km radius in the Setúbal Peninsula, 20 km south of Lisbon. International wood trade, with its political as well as economic ramifications, has been seriously jeopardized. The origin of the population of PWN found in Portugal remains elusive. Several hypotheses may be considered regarding pathway analysis, basically from two general origins: North America or the Far East (Japan or China). World trade of wood products such as timber, wooden crates, palettes, etc… play an important role in the potential dissemination of the pinewood nematode. In fact, human activities involving the movement of wood products may be considered the single most important factor in spreading of the PWN. Despite the dedicated and concerted actions of government agencies, this disease continues to spread. Very recently (2006), in Portugal, forestry and phytosanitary authorities (DGRF and DGPC) have announced a new strategy for the control and ultimately the erradication of the nematode, under the coordination of the national program for the control of the pinewood nematode (PROLUNP). Research regarding the bioecology of the nematode and insect as well as new detection methods, e.g., involving real-time PCR, has progressed since 1999. International agreements (GATT, WTO) and sharing of scientific information is of paramount importance to effectively control the nematode and its vector, and thus protect our forest ecosystems and forest economy.

Progress in the use of scanning electron microscopy (SEM) applied to the taxonomy of the genus Bursaphelenchus (Nematoda: Parasitaphelenchidae)

The scanning electron microscope (SEM) has been a major tool in detailed morphological observations of plant parasitic nematodes during the last 30 years, efficiently complementing light microscopical (LM) studies. Nematodes are extremely difficult to observe and characterize due to their small size (aprox. 1 mm long) and paucity of morphological characters, so detailed surface observations of several organs and nematode regions are of the highest value. Among plant parasitic nematodes, one of the most devastating species is the “pinewood nematode” (PWN), Bursaphelenchus xylophilus, which has been a major problem for forest species, and in particular pines, in Asia (Japan, China, Korea) and has been recently detected in the European Union (Portugal). B. xylophilus belongs to a closely related, morphologically similar group of species, within the genus Bursaphelenchus, and designated by the “xylophilus group”. SEM has become a crucial tool in observing several genital characters of males and females, such as male genital papillae, male copulatory spicules, female vulval flap and female genital papillae.s In this presentation, we will show how SEM has been utilized to observe and characterize the shape of the vulval flap, the presence/ absence of papillae near the flap, and confirm the presence and the arrangement of the male genital papillae. LM is also used in this work to show its value as a complementary tool to SEM, in both genital characteristics and other, general, characters of the genus Bursaphelenchus, such as the male bursa and cephalic region.

Pine wilt disease: global issues, trade and economic impact

Pine wilt disease (PWD) is perhaps the most serious threat to pine forests worldwide. Since it´s discovery in the early XXth century by Japanese forest researchers, and the relationship with its causative agent, the pinewood nematode (PWN) Bursaphelenchus xylophilus, in the 1970s, PWD has wreaked havoc wherever it appears. Firstly in the Far East (Japan, China and Korea) and now, more recently in 1999, in the EU (Portugal). The forest sector in Portugal plays a major role in the Portuguese economy with a 12% contribution to the industrial gross domestic product, 3.2% of the gross domestic product, 10% of foreign trade and 5% of national employment. Maritime pine (Pinus pinaster) is one of the most important pine productions, and industrial activity, such as the production of wood and resin, as well as coastal protection associated with sand dunes. Also, stone pine (Pinus pinea) plays an important role in the economy with a share derived from the exports of high-quality pineon seed. Thus, the tremendous economical and ecological impact of the introduction of a pest and pathogen such as the PWN, although as far as is known, the only species susceptible to the nematode is maritime pine. Immediately following detection, the research team involved (Univ. Évora, INIAP) informed the national plant quarantine and forest authorities, which relayed the information to Brussels and the appropriate EU authorities. A task force (GANP), followed by a national program (PROLUNP) was established. Since then, national surveys have been taking place, involving MADRP (Ministry of Agriculture), the University of Évora and several private corporations (e.g. UNAC). Forest growers in the area are particularly interested and involved since the area owned by the growers organizations totals 700 000 ha, largely affected by PWD. Detection of the disease has led to serious consequences and restrictions regarding exploration and commercialization of wood. A precautionary phytosanitary strip, 3 km-wide, has been recently (2007) established surrounding the affected area. The Portuguese government, through its national program PROLUNP, has been deeply involved since 1999, and in conjunction with the EU (Permanent Phytosanitary Committee, and FVO) and committed to controlling this nematode and the potential spread to the rest of the country and to the rest of the EU. The global impact of the presence of Bursaphelenchus xylophilus or the threat of its introduction and the resulting pine wilt disease in forested areas in different parts of the world is of increasing concern economically. The concern is exacerbated by the prevailing debate on climate change and the putative impact this could have on the vulnerability of the world’s pine forests to this disease. The scientific and regulatory approach taken in different jurisdictions to the threat of pine wilt disease varies from country to country depending on the perceived vulnerability of their pine forests to the disease and/or to the economic cost due to lost trade in wood products. Much of the research surrounding pine wilt disease has been located in the northern hemisphere, especially in southern Europe and in the warmer, coastal, Asian countries. However, there is an increased focus on this problem also in those countries in the southern hemisphere where plantations of susceptible pine have been established over the years. The forestry sector in Australia and New Zealand are on “high alert” for this disease and are practicing strict quarantine procedures at all ports of entry for wood products. As well, there is heightened awareness, as there is worldwide, for the need to monitor wood packaging materials for all imported goods. In carrying out the necessary monitoring and assessment of products for B. xylophilus and its vectors substantial costs are incurred especially when decisions have to be made rapidly and regardless of whether the outcome is positive or negative. Australia’s response recently to the appearance of some dying pines in a plantation illustrated the high sensitivity of some countries to this disease. Some $200,000 was spent on the assessment in order to save a potential loss of millions of dollars to the disease. This rapid, co-ordinated response to the report was for naught, because once identified it was found not to be B. xylophilus. This illustrates the particular importance of taking the responsibility at all levels of management to secure the site and the need of a rapid, reliable diagnostic method for small nematode samples for use in the field. Australia is particularly concerned about the vulnerability of its 1million hectares of planted forests, 80% of which are Pinus species, to attack from incursions of one or more species of the insect vector. Monochamus alternatus incursions in wood pallets have been reported from Brisbane, Queensland. The climate of this part of Australia is such that the Pinus plantations are particularly vulnerable to the potential outcome of such incursions, and the state of Queensland is developing a risk management strategy and a proactive breeding programme in response to this putative threat. New Zealand has 1.6 million hectares of planted forests and 89% of the commercial forest is Pinus radiata. Although the climate where these forests are located tends to be somewhat cooler than that in Australia the potential for establishment and development of the disease in that country is believed to be high. The passage alone of 200,000 m³/year of wood packaging through New Zealand ports is itself sufficient to require response. The potential incursion of insect vectors of pinewood nematode through the port system is regarded as high and is monitored carefully. The enormous expansion of global trade and the continued use of unprocessed/inadequately-processed wood for packaging purposes is a challenge for all trading nations as such wood packaging material often harbours disease or pest species. The extent of this problem is readily illustrated by the expanding economies and exports of countries in south-east Asia. China. Japan and Korea have significant areas of forestland infested with B. xylophilus. These countries too are among the largest exporting countries of manufactured goods. Despite the attempts of authorities to ensure that only properly treated wood is used in the crating and packaging of goods B. xylophilus and/or its insect vector infested materials is being recorded at ports worldwide. This reminds us, therefore, of the ease with which this nematode pest can gain access to forest lands in new geographic locations through inappropriate use, treatment or monitoring of wood products. It especially highlights the necessity to find an alternative to using low-grade lumber for packaging purposes. Lest we should believe that all wood products are always carriers of B. xylophilus and its vectors, it should be remembered that international trade of all kinds has occurred for thousands of years and that lumber-born pests and diseases do not have worldwide distribution. Other physico-biological factors have a significant role in the occurrence, establishment and sustainability of a disease. The question is often raised as to why the whole of southern Europe doesn’t already have B. xylophilus and pine wilt disease. European countries have traded with countries that are infested with B. xylophilus for hundreds of years. Turkey is an example of a country that appears to be highly vulnerable to pine wilt disease due to its extensive forests in the warm, southern region where the vector, Monochamus galloprovincialis, occurs. However, there is no record of the presence of B. xylophilus occurring there despite the importation of substantial quantities of wood from several countries In many respects, Portugal illustrates both the challenge and the dilemma. In recent times B. xylophilus was discovered there in the warm coastal region. The research, administrative and quarantine authorities responded rapidly and B. xylophilus appears to have been confined to the region in which it was found. The rapid response would seem to have “saved the day” for Portugal. Nevertheless, it raises again the long-standing questions, how long had B. xylophilus been in Portugal before it was found? If Lisbon was the port of entry, which seems very likely, why had B. xylophilus not entered Lisbon many years earlier and established populations and the pine wilt disease? Will the infestation in Portugal be sustainable and will it spread or will it die out within a few years? We still do not have sufficient understanding of the biology of this pest to know the answers to these questions.

Pine Wilt Disease: A Threat to Pine Forests in Turkey?

The pinewood nematode, is the causal agent of pine wilt disease, a serious threat to native pine forest in eastern Asia (Japan, Korea, China and Taiwan) and some parts of North America (USA, Canada and Mexico). In 1999, this nematode was found and identified for the first time in Portugal and in Europe. The detection of this quarantine pest in Portugal has indicated the need to know more about the distribution of Bursaphelenchus spp. in coniferous trees in Europe in order to describe the geographic range of the species and to act quickly in case of the nematode’s unwanted introduction into other European regions. Pine forest has a wide distribution in Turkey that increases the number of susceptible host trees for pinewood nematode. Because of these resaons, some regions of Turkey were surveyed for the presence of the nematode. Three different species of Bursaphelenchus were found. However, B. xylophilus was not detected. The detection of B. mucronatus, very similar to B. xylophilus biologically and morphologically, is very important. The presence of this species indicates that B. xylophilus could spread easly in conifer forests of Turkey. A study was conducted to determine the pathogenicity of B. mucronatus and 80% of seedlings of P. sylvestris were wilted. Biological characteristics of M. galloprovincialis were compared with M. carolinensis, Nort American vector, and some of them were found to be similar.

Developmental biology and cytogenetics of B. xylophilus

The pine wood nematode Bursaphelenchus xylophilus reproduces bisexually: a haploid sperm fertilizes a haploid oocyte, and the two pronuclei rearrange, move together, fuse, and begin diploid development. Early embryonic events taking place in the B. xylophilus embryo are similar to those of Caenorhabditis elegans, although the anterior-posterior axis appeares to be determined oppositely to that observed for C. elegans. Thai is, in the B. xylophilus embryo, the male pronucleus emerges at the future anterior end, whereas the female pronucleus appeares laterally. To understand the evolution of nematode developmental systems, we cloned the full length of Bx-tbb-1 (beta tubulin) from B. xylophilus cDNA and attempted to apply reverse genetics analysis to B. xylophilus. Several lengths of double stranded RNA (dsRNA) for the Bx-tbb-1 gene were synthesized by in vitro transcription, and both B. xylophilus and C. elegans were soaked in dsRNA for RNAi. Both nematodes could suck up the dsRNA, and we could detect the abnormal phenotypes caused by Bx-tbb-1 dsRNA in C. elegans, but not in B. xylophilus. We suspect that systemic RNAi might be suppressed in B. xylophilus and are attempting to establish other methods for functionally analyzing B. xylophilus genes.

Taxonomic databases for Bursaphelenchus and other aphelenchoid nematodes

Nematodes are the most abundant metazoans, comprising more than 80% of all animals alive today. Since 1743, when Needham (Needham, 1743) described the first nematode, approximately 20,000 - 30,000 species have been named, with estimates of species remaining to be described ranging from 100,000 to 1 million (Blaxter, 2004; De Ley, 2000). Unfortunately, the taxonomic community is woefully inadequate for this task. The number of taxonomists currently describing new species of nematodes around the world is less than 100, and significant increases are not expected. If each of these taxonomists were able to describe 10 new species every year, it would take between 100 to 1,000 years to name these yet to be described species.