Predicting future distributions of mountain plants under climate change: does dispersal capacity matter?

Many studies have investigated the potential impacts of climate change on the distribution of plant species, but few have attempted to constrain projections through plant dispersal limitations. Instead, most studies published so far have simplified dispersal as either unlimited or null. However, depending on the dispersal capacity of a species, landscape fragmentation, and the rate of climatic change, these assumptions can lead to serious over- or underestimation of the future distribution of plant species.
To quantify the discrepancies between simulations accounting for dispersal or not, we carried out projections of future distribution over the 21st century for 287 mountain plant species in a study area of the western Swiss Alps. For each species, simulations were run for four dispersal scenarios (unlimited dispersal, no dispersal, realistic dispersal, and realistic dispersal with long-distance dispersal events) and under four climate change scenarios.
Although simulations accounting for realistic dispersal limitations did significantly differ from those considering dispersal as unlimited or null in terms of projected future distribution, the unlimited dispersal simplification did nevertheless provide good approximations for species extinctions under more moderate climate change scenarios. Overall, simulations accounting for dispersal limitations produced, for our mountainous study area, results that were significantly closer to unlimited dispersal than to no dispersal. Finally, analysis of the temporal pattern of species extinctions over the entire 21st century revealed that important species extinctions for our study area might not occur before the 2080–2100 period, due to the possibility of a large number of species shifting their distribution to higher elevation.     

Synthesis and biological evaluation of new heterocyclic quinolinones as anti-parasite and anti-HIV drug candidates

We have synthesized quinolinones with potential antiparasitic and anti-HIV activities by an original two-step method involving microwave irradiation and have evaluated their activities against Plasmodium falciparum, Leishmania donovani, Trichomonas vaginalis, and HIV. None of the tested compounds had been previously described using this method of synthesis. One of the compounds had interesting antiparasitic and anti-HIV activity, which could be improved by substitution with different radicals.     

Gene expression profiling of single bovine embryos uncovers significant effects of in vitro maturation, fertilization and culture

In vitro production (IVP) has been shown to affect embryonic gene expression and often result in large offspring syndrome (LOS) in cattle and sheep. To dissect the effects of in vitro maturation, fertilization and culture on bovine embryos, we compared the expression profiles of single blastocysts generated by: (1) in vitro maturation, fertilization and culture (IVF); (2) in vivo maturation, fertilization and in vitro culture (IVD); and (3) in vivo maturation, fertilization and development (AI). To conduct expression profiling, total RNA was isolated from individual embryos, linearly amplified and hybridized to a custom bovine cDNA microarray containing approximately 6,300 unique genes. There were 306, 367, and 200 genes differentially expressed between the AI and IVD, IVF and IVD, and AI and IVF comparisons, respectively. Interestingly, 44 differentially expressed genes were identified between the AI embryos and both the IVF and IVD embryos, making these potential candidates for LOS. There were 60 genes differentially expressed between the IVF embryos and the AI and IVD embryos. The Gene Ontology category "RNA processing" was over-represented among the genes that were down-regulated in the IVF embryos, indicating an effect of in vitro oocyte maturation/fertilization on the ability to transcribe maternal RNA stores. A culture effect on the expression of genes involved in translation was also observed by the comparison of AI with IVD embryos.     

SYMBIODINIUM (DINOPHYTA) DIVERSITY AND STABILITY IN AQUARIUM CORALS1

Indo‐Pacific reef corals growing for years in closed‐system aquaria provide an alternate means to investigate host–symbiont specificity and stability. The diversity of dinoflagellate endosymbionts (Symbiodinium spp.) from coral communities in private and public aquaria was investigated using molecular‐genetic analyses. Of the 29 symbiont types (i.e., species) identified, 90% belonged to the most prevalent group of Symbiodinium harbored by Indo‐Pacific reef corals, Clade C, while the rest belonged to Clade D. Sixty‐five percent of all types were known from field surveys conducted throughout the Pacific and Indian oceans. Because specific coral–dinoflagellate partnerships appear to have defined geographic distributions, correspondence of the same symbionts in aquarium and field‐collected specimens identifies regions where particular colonies must have been collected in the wild. Symbiodinium spp. in clade D, believed to be “stress‐tolerant” and/or “opportunistic,” occurred in a limited number of individual colonies. The absence of a prevalent, or “weedy,” symbiont suggests that conditions under which aquarium corals are grown do not favor competitive replacements of their native symbiont populations. The finding of typical and diverse assemblages of Symbiodinium spp. among aquarium corals living many years under variable chemical/physical conditions, artificial and natural light, while undergoing fragmentation periodically, indicates that individual colonies maintain stable, long‐term symbiotic associations.     

E4orf1 Limits the Oncolytic Potential of the E1B-55K Deletion Mutant Adenovirus

Clinical trials have shown oncolytic adenoviruses to be tumor selective with minimal toxicity toward normal tissue. The virus ONYX-015, in which the gene encoding the early region 1B 55-kDa (E1B-55K) protein is deleted, has been most effective when used in combination with either chemotherapy or radiation therapy. Therefore, improving the oncolytic nature of tumor-selective adenoviruses remains an important objective for improving this form of cancer therapy. Cells infected during the G₁ phase of the cell cycle with the E1B-55K deletion mutant virus exhibit a reduced rate of viral late protein synthesis, produce fewer viral progeny, and are less efficiently killed than cells infected during the S phase. Here we demonstrate that the G₁ restriction imposed on the E1B-55K deletion mutant virus is due to the viral oncogene encoded by open reading frame 1 of early region 4 (E4orf1). E4orf1 has been reported to signal through the phosphatidylinositol 3′-kinase pathway leading to the activation of Akt, mTOR, and p70 S6K. Evidence presented here shows that E4orf1 may also induce the phosphorylation of Akt and p70 S6K in a manner that depends on Rac1 and its guanine nucleotide exchange factor Tiam1. Accordingly, agents that have been reported to disrupt the Tiam1-Rac1 interaction or to prevent phosphorylation of the ribosomal S6 kinase partially alleviated the E4orf1 restriction to late viral protein synthesis and enhanced tumor cell killing by the E1B-55K mutant virus. These results demonstrate that E4orf1 limits the oncolytic nature of a conditionally replicating adenovirus such as ONYX-015. The therapeutic value of similar oncolytic adenoviruses may be improved by abrogating E4orf1 function.Conditionally replicating adenoviruses are a novel class of biological agents used to treat cancer (57). The E1B-55K deletion mutant virus ONYX-015, originally known as dl1520 (4), is one of the first of such agents (7). H101 is another E1B-55K deletion mutant adenovirus that is being used for tumor therapy in China (30, 78). We previously reported that cells infected during the G₁ phase of the cell cycle with E1B-55K deletion mutant adenoviruses exhibit a reduced rate of viral late protein synthesis, produce fewer viral progeny, and are less effectively killed than cells infected during S phase (34, 35, 66). These observations indicated that the E1B-55K deletion mutant virus ONYX-015 is restricted in cells infected in G₁. This restriction is significant because a large fraction of cells within a tumor exist in the G₁ phase of the cell cycle (71). Here we show that the G₁ restriction imposed on the E1B-55K deletion mutant virus is due to the viral oncogene encoded by open reading frame 1 of early region 4 (E4orf1).The E4orf1-encoded protein is a small adapter molecule that associates with PDZ domain-containing proteins including MUPP1, PATJ, MAGI-1, ZO-2, and Dlg1 (46). PDZ domain-containing proteins often serve as scaffolds for the assembly of signaling complexes at the plasma membrane (64). Through its association with PDZ domain-containing proteins, the E4orf1-encoded protein promotes signaling through the phosphatidylinositol 3′-kinase (PI3-kinase) pathway to effectors such as protein kinase B (Akt), the mammalian target of rapamycin (mTOR), and the S6 ribosomal protein kinase (p70 S6K) (27, 54). Through these effectors, PI3-kinase alters protein synthesis and cell survival (21, 28). E4orf1 is the principal oncogenic determinant of species D adenovirus type 9 (42). The transforming ability of E4orf1 can be blocked by the PI3-kinase inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY249002","term_id":"1257710161","term_text":"LY249002"}}LY249002 (27). However, phosphorylation of p70 S6K can also proceed by pathways that are independent of PI3-kinase or Akt. For example, the Rho-like GTPase Rac1 can activate p70 S6K (17). Rac1 is itself regulated by cellular factors to which it binds, including the Rac1-specific guanine nucleotide exchange factor T-cell lymphoma invasion and metastasis 1 protein (Tiam1). Tiam1 and the neural tissue-associated F-actin-binding protein neurabin II or spinophilin recruit p70 S6K into a complex containing Rac1, resulting in increased phosphorylation of p70 S6K (12, 36, 50). Interestingly, both Tiam1 and neurabin II are PDZ-containing proteins. These observations provided a potential basis by which E4orf1 may modulate protein synthesis and cell survival.In this report, we show for the first time that E4orf1 restricts the abilities of the E1B-55K deletion mutant virus to produce viral progeny, to direct viral late protein synthesis, and to kill tumor cells. Drugs that are reported to prevent phosphorylation of p70 S6K or to disrupt the interaction between Tiam1 and Rac1 increase the cell-killing ability of the E1B-55K deletion mutant virus to nearly the same level observed for an E1B-55K/E4orf1 double mutant and the wild-type virus. By uncovering a role for E4orf1 in the course of a lytic adenovirus infection, this study presents novel genetic and pharmacological means by which the effectiveness of replicating oncolytic adenoviruses can be improved.     

Deficiency of Niemann-Pick Type C-1 Protein Impairs Release of Human Immunodeficiency Virus Type 1 and Results in Gag Accumulation in Late Endosomal/Lysosomal Compartments

Human immunodeficiency virus type 1 (HIV-1) relies on cholesterol-laden lipid raft membrane microdomains for entry into and egress out of susceptible cells. In the present study, we examine the need for intracellular cholesterol trafficking pathways with respect to HIV-1 biogenesis using Niemann-Pick type C-1 (NPC1)-deficient (NPCD) cells, wherein these pathways are severely compromised, causing massive accumulation of cholesterol in late endosomal/lysosomal (LE/L) compartments. We have found that induction of an NPC disease-like phenotype through treatment of various cell types with the commonly used hydrophobic amine drug U18666A resulted in profound suppression of HIV-1 release. Further, NPCD Epstein-Barr virus-transformed B lymphocytes and fibroblasts from patients with NPC disease infected with a CD4-independent strain of HIV-1 or transfected with an HIV-1 proviral clone, respectively, replicated HIV-1 poorly compared to normal cells. Infection of the NPCD fibroblasts with a vesicular stomatitis virus G-pseudotyped strain of HIV-1 produced similar results, suggesting a postentry block to HIV-1 replication in these cells. Examination of these cells using confocal microscopy showed an accumulation and stabilization of Gag in LE/L compartments. Additionally, normal HIV-1 production could be restored in NPCD cells upon expression of a functional NPC1 protein, and overexpression of NPC1 increased HIV-1 release. Taken together, our findings demonstrate that intact intracellular cholesterol trafficking pathways mediated by NPC1 are needed for efficient HIV-1 production.Human immunodeficiency virus type 1 (HIV-1) is a complex retrovirus highly dependent upon a myriad of cellular mechanisms for successful virus replication. Cholesterol plays a pivotal role throughout the HIV-1 life cycle (23, 40, 41, 64). HIV-1 entry, assembly, and budding processes occur at cholesterol-enriched membrane microdomains known as lipid rafts, and depletion of cellular cholesterol markedly and specifically reduces HIV-1 particle production. Virion-associated cholesterol is required for fusion and subsequent infection of susceptible cells (41), and cholesterol-sequestering drugs, such as β-cyclodextrin, render the virus incompetent for cell entry (4, 25, 57). Therefore, intracellular cholesterol trafficking pathways that allow nascent HIV-1 particles to acquire lipids appear critical for virus replication.Recent evidence supports a critical role for cholesterol trafficking and homeostasis in viral replication, showing that the HIV-1 accessory protein Nef increases synthesis and transport of cholesterol to both lipid rafts and progeny virions and induces multiple genes involved in cholesterol synthesis (80, 88). More recent studies have revealed that binding of Nef to the ATP-binding cassette transporter A1 (ABCA1) leads to impairment of ABCA1-dependent cholesterol efflux and an accumulation of lipids within the cell (51).Mammalian cells acquire cholesterol primarily from endocytosed low-density lipoproteins (LDL). The Niemann-Pick type C-1 (NPC1) protein is well known for its role in intracellular trafficking of LDL-derived free unesterified cholesterol. Dysfunctional NPC1 activity leads to development of NPC disease, a rare, autosomal recessive, neurodegenerative disorder characterized by the massive accumulation of cholesterol and glycosphingolipids in late endosomal/lysosomal (LE/L) compartments (61). In normal cells, endocytosed LDLs are delivered to the LE/Ls, where they are hydrolyzed and free cholesterol is released. Homeostasis is achieved when cholesterol is then rapidly transported out of the LE/Ls to the plasma membrane and endoplasmic reticulum (ER) (17, 19, 42, 73, 85), or first to the trans-Golgi (TG) network (TGN) and then to the ER (76). In NPC1-deficient (NPCD) cells, the cholesterol does not exit the endocytic pathway, resulting in its accumulation within LE/L structures.In 95% of NPC patients, the disease is caused by mutations in the NPC1 gene, while the remaining 5% harbor mutations in the NPC2 gene (50, 72, 79). One of the most frequently found and extensively characterized NPC1 mutations is the I1061T mutation (37, 38, 86). This mutation results in misfolding of the NPC1 protein, leading to its degradation and causing an 85% decrease in cellular NPC1 expression (20). Cells with such low levels of functional NPC1 maintain only 38% of normal sphingomyelinase activity and have impaired cholesterol esterification and trafficking.NPC1 is a large, multispanning protein that resides in the limiting membrane of the LE and binds cholesterol via its N-terminal domain (31). While the complete physiological function of NPC1 is still unclear, NPC1 does share homology with the resistance-nodulation-division family of prokaryotic permeases and may function as a transmembrane efflux pump to transport cargos in LEs (9, 75). Other studies suggest that NPC1 might also function in vesicle-mediated pathways for cargo transportation from LEs to other intracellular sites (21, 33). Recent studies by Infante et al. have propelled forward our understanding of how NPC1 works together with NPC2, also known to bind cholesterol, to support cholesterol efflux from the LE (32). Their findings provide a basis for either of two possible models, with respect to cholesterol trafficking: (i) NPC1 binds cholesterol found within the LE and mediates either direct export or transfer to NPC2 for delivery to a cholesterol efflux transporter, such as ABCA1; or (ii) NPC2 is the first to bind cholesterol and then mediate its delivery to NPC1 for direct export or transfer to ABCA1. These recent findings underscore the highly critical role of these proteins in maintaining intracellular cholesterol homeostasis.In addition to its role in sterol trafficking, some studies suggest that the NPC pathway may be directly involved in trafficking multiple proteins from LE/L compartments. LEs act as sorting stations to deliver endocytosed molecules to L''s for degradation, while at the same time retrieving other classes of proteins and lipids for transport back to nondegradative compartments (3, 14, 15, 28, 63, 69, 78). LE compartments also serve as sorting stations for HIV-1 viral proteins and represent a major site for HIV-1 assembly and budding (7, 12, 16, 22, 24, 57, 59).The endosomal trafficking defects observed in NPCD cells extend to proteins such as IGF2/MPR, NPC1, and annexin II, all of which utilize the endosomal recycling pathway (42, 74). Electron microscopy studies have shown that within the LEs of NPCD cells these proteins are trapped in the cholesterol-enriched membrane-bound vesicular structures (47). Cholesterol and glycosphingolipid accumulation within NPCD cells appears to disrupt Rab9 GTPase function in LE-to-TGN transport, trapping Rab9-associated proteins, such as vimentin, Tip47, and the mannose-6-phosphate receptor in LEs (18, 83). Overexpression of Rab7 and Rab9 GTPases can reverse the cholesterol accumulation phenotype caused by NPCD (8, 84). These observations suggest that NPC1, directly or indirectly, plays a role in protein export from LEs. It is unknown whether NPC1 is involved in the export of HIV-1 proteins from LEs; however, the Rab9 GTPase-mediated pathway is known to be required for HIV-1 replication (53). This strongly suggests that HIV assembly will be hindered when the NPC pathway is disrupted.Given the function of NPC1 in mediating intracellular cholesterol trafficking within the LE and given the need of HIV-1 for cholesterol, NPC1 involvement in HIV-1 biogenesis is highly likely. In the present study, using cells treated with U18666A or NPCD cells, we show that impaired NPC1 function results in profound suppression of HIV-1 replication. Further, our findings demonstrate that the NPC1 protein is essential for proper trafficking of the HIV-1 Gag protein during the late stages of assembly and budding. It appears that in NPCD cells, in which cholesterol and cellular proteins accumulate in LE/L compartments, the viral Gag protein fails to traffic properly and accumulates within these compartments, resulting in decreased particle production. Our findings not only reinforce the dependence of HIV-1 on cholesterol homeostasis but also support a role for NPC1 in HIV-1 viral protein trafficking and particle release from infected cells.     

3-Hydroxypropionyl-Coenzyme A Dehydratase and Acryloyl-Coenzyme A Reductase,Enzymes of the Autotrophic 3-Hydroxypropionate/4-Hydroxybutyrate Cycle in the Sulfolobales

A 3-hydroxypropionate/4-hydroxybutyrate cycle operates in autotrophic CO₂ fixation in various Crenarchaea, as studied in some detail in Metallosphaera sedula. This cycle and the autotrophic 3-hydroxypropionate cycle in Chloroflexus aurantiacus have in common the conversion of acetyl-coenzyme A (CoA) and two bicarbonates via 3-hydroxypropionate to succinyl-CoA. Both cycles require the reductive conversion of 3-hydroxypropionate to propionyl-CoA. In M. sedula the reaction sequence is catalyzed by three enzymes. The first enzyme, 3-hydroxypropionyl-CoA synthetase, catalyzes the CoA- and MgATP-dependent formation of 3-hydroxypropionyl-CoA. The next two enzymes were purified from M. sedula or Sulfolobus tokodaii and studied. 3-Hydroxypropionyl-CoA dehydratase, a member of the enoyl-CoA hydratase family, eliminates water from 3-hydroxypropionyl-CoA to form acryloyl-CoA. Acryloyl-CoA reductase, a member of the zinc-containing alcohol dehydrogenase family, reduces acryloyl-CoA with NADPH to propionyl-CoA. Genes highly similar to the Metallosphaera CoA synthetase, dehydratase, and reductase genes were found in autotrophic members of the Sulfolobales. The encoded enzymes are only distantly related to the respective three enzyme domains of propionyl-CoA synthase from C. aurantiacus, where this trifunctional enzyme catalyzes all three reactions. This indicates that the autotrophic carbon fixation cycles in Chloroflexus and in the Sulfolobales evolved independently and that different genes/enzymes have been recruited in the two lineages that catalyze the same kinds of reactions.In the thermoacidophilic autotrophic crenarchaeum Metallosphaera sedula, CO₂ fixation proceeds via a 3-hydroxypropionate/4-hydroxybutyrate cycle (8, 23, 24, 28) (Fig. ​(Fig.1).1). A similar cycle may operate in other autotrophic members of the Sulfolobales and in mesophilic Crenarchaea (Cenarchaeum sp. and Nitrosopumilus sp.) of marine group I. The cycle uses elements of the 3-hydroxypropionate cycle that was originally discovered in the phototrophic bacterium Chloroflexus aurantiacus (11, 16, 17, 19, 20, 32, 33). It involves the carboxylation of acetyl-coenzyme A (CoA) to malonyl-CoA by the biotin-dependent acetyl-CoA carboxylase. Malonyl-CoA is reduced via malonate semialdehyde to 3-hydroxypropionate (1), which is further reductively converted to propionyl-CoA (3). Propionyl-CoA is carboxylated to (S)-methylmalonyl-CoA by a propionyl-CoA carboxylase that is similar or identical to acetyl-CoA carboxylase. In fact, only one copy of the genes for the acetyl-CoA/propionyl-CoA carboxylase subunits is present in most Archaea, suggesting that this is a promiscuous enzyme that acts on both acetyl-CoA and propionyl-CoA (24). (S)-Methylmalonyl-CoA is epimerized to (R)-methylmalonyl-CoA, followed by carbon rearrangement to succinyl-CoA by coenzyme B₁₂-dependent methylmalonyl-CoA mutase.Open in a separate windowFIG. 1.Proposed 3-hydroxypropionate/4-hydroxybutyrate cycle in M. sedula and other members of the Sulfolobales. Enzymes are the following: 1, acetyl-CoA carboxylase; 2, malonyl-CoA reductase (NADPH); 3, malonate semialdehyde reductase (NADPH); 4, 3-hydroxypropionyl-CoA synthetase (3-hydroxypropionate-CoA ligase, AMP forming); 5, 3-hydroxypropionyl-CoA dehydratase; 6, acryloyl-CoA reductase (NADPH); 7, propionyl-CoA carboxylase; 8, methylmalonyl-CoA epimerase; 9, methylmalonyl-CoA mutase; 10, succinyl-CoA reductase (NADPH); 11, succinate semialdehyde reductase (NADPH); 12, 4-hydroxybutyryl-CoA synthetase (4-hydroxybutyrate-CoA ligase, AMP-forming); 13, 4-hydroxybutyryl-CoA dehydratase; 14, crotonyl-CoA hydratase; 15, (S)-3-hydroxybutyryl-CoA dehydrogenase (NAD⁺); 16, acetoacetyl-CoA β-ketothiolase. The two steps of interest are highlighted.In Chloroflexus succinyl-CoA is converted to (S)-malyl-CoA, which is cleaved by (S)-malyl-CoA lyase to acetyl-CoA (thus regenerating the CO₂ acceptor molecule) and glyoxylate (16). Glyoxylate is assimilated into cell material by a yet not completely resolved pathway (37). In Metallosphaera succinyl-CoA is converted via 4-hydroxybutyrate to two molecules of acetyl-CoA (8), thus regenerating the starting CO₂ acceptor molecule and releasing another acetyl-CoA for biosynthesis. Hence, the 3-hydroxypropionate/4-hydroxybutyrate cycle (Fig. ​(Fig.1)1) can be divided into two parts. The first part transforms one acetyl-CoA and two bicarbonates into succinyl-CoA, and the second part converts succinyl-CoA to two acetyl-CoA molecules.The reductive conversion of 3-hydroxypropionate to propionyl-CoA requires three enzymatic steps: activation of 3-hydroxypropionate to its CoA ester, dehydration of 3-hydroxypropionyl-CoA to acryloyl-CoA, and reduction of acryloyl-CoA to propionyl-CoA. In C. aurantiacus these three steps are catalyzed by a single large trifunctional enzyme, propionyl-CoA synthase (2). This 200-kDa fusion protein consists of a CoA ligase, a dehydratase, and a reductase domain. Attempts to isolate a similar enzyme from M. sedula failed. Rather, a 3-hydroxypropionyl-CoA synthetase was found (3), suggesting that the other two reactions may also be catalyzed by individual enzymes.Here, we purified the missing enzymes 3-hydroxypropionyl-CoA dehydratase and acryloyl-CoA reductase from M. sedula, identified the coding genes in the genome of M. sedula and other members of the Sulfolobales, produced recombinant enzymes as proof of function, and studied the enzymes in some detail. A comparison with the respective domains of propionyl-CoA synthase from C. aurantiacus indicates that the conversion of 3-hydroxypropionate to propionyl-CoA via the 3-hydroxypropionate route has evolved independently in these two phyla.     

A combined microscopic-molecular method for the diagnosis of strongylid infections in sheep

We evaluated a combined microscopic-molecular approach for the diagnosis of key strongylid infections in sheep using panels of well-defined control and test samples. The method established is based on the separation of nematode eggs from faecal samples using a salt flotation procedure, the extraction and column-purification of genomic DNA, followed by real-time PCR and melting-curve analysis. Specific and semi-quantitative amplification from (a minimum of 0.1-2.0 pg) genomic DNA of Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus spp., Cooperia oncophora, Oesophagostomum columbianum, Oesophagostomum venulosum or Chabertia ovina is achieved using a specific, forward oligonucleotide primer located in the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA (rDNA) together with a conserved reverse primer in the large subunit of rDNA. Using a panel of well-defined genomic DNA samples from eggs from sheep monospecifically infected with H. contortus or Te. circumcincta, there was a correlation between cycle threshold (Ct) values in the PCR and numbers of egg per gram of faeces, thus allowing the semi-quantitation of parasite DNA in faeces. The findings of the present study indicate that a microscopic-molecular approach provides a useful tool for diagnosis, for epidemiological and ecological surveys as well as for integration into parasite monitoring, drug resistance (i.e. ‘egg count reduction’) testing or control programmes, particularly following semi- or full-automation.