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• W2621590622 abstract "The salmon louse, Lepeophtheirus salmonis, is an endemic ectoparasite on salmonid fish that is challenging for the salmon farming industry and wild fish. Salmon lice produce high numbers of offspring, necessitating sequestration of large amounts of lipids into growing oocytes as a major energy source for larvae, most probably mediated by lipoproteins. The microsomal triglyceride transfer protein (MTP) is essential for the assembly of lipoproteins. Salmon lice have three L. salmonis MTP (LsMTP) transcript variants encoding two different protein isoforms, which are predicted to contain three β-sheets (N, C, and A) and a central helical domain, similar to MTPs from other species. In adult females, the LsMTPs are differently transcribed in the sub-cuticular tissues, the intestine, the ovary, and in the mature eggs. RNA interference-mediated knockdown of LsMTP in mature females gave offspring with significantly fewer neutral lipids in their yolk and only 10–30% survival. The present study suggests the importance of LsMTP in reproduction and lipid metabolism in adult female L. salmonis, a possible metabolic bottleneck that could be exploited for the development of new anti-parasitic treatment methods. The salmon louse, Lepeophtheirus salmonis, is an endemic ectoparasite on salmonid fish that is challenging for the salmon farming industry and wild fish. Salmon lice produce high numbers of offspring, necessitating sequestration of large amounts of lipids into growing oocytes as a major energy source for larvae, most probably mediated by lipoproteins. The microsomal triglyceride transfer protein (MTP) is essential for the assembly of lipoproteins. Salmon lice have three L. salmonis MTP (LsMTP) transcript variants encoding two different protein isoforms, which are predicted to contain three β-sheets (N, C, and A) and a central helical domain, similar to MTPs from other species. In adult females, the LsMTPs are differently transcribed in the sub-cuticular tissues, the intestine, the ovary, and in the mature eggs. RNA interference-mediated knockdown of LsMTP in mature females gave offspring with significantly fewer neutral lipids in their yolk and only 10–30% survival. The present study suggests the importance of LsMTP in reproduction and lipid metabolism in adult female L. salmonis, a possible metabolic bottleneck that could be exploited for the development of new anti-parasitic treatment methods. The microsomal triglyceride transfer protein (MTP) was first reported as an endoplasmic reticulum resident protein that catalyzes the transfer of neutral lipids between membranes (1.Wetterau J.R. Zilversmit D.B. A triglyceride and cholesteryl ester transfer protein associated with liver microsomes.J. Biol. Chem. 1984; 259: 10863-10866Abstract Full Text PDF PubMed Google Scholar). Later it was found that MTP is also essential for the synthesis and secretion of lipoproteins containing apoB (2.Wetterau J.R. Lin M.C. Jamil H. Microsomal triglyceride transfer protein.Biochim. Biophys. Acta. 1997; 1345: 136-150Crossref PubMed Scopus (286) Google Scholar). MTP belongs to the large lipid transfer protein superfamily and it functions as a transporter of lipids in the assembly of nascent lipoprotein particles within the endoplasmic reticulum (3.Jamil H. Dickson Jr., J.K. Chu C.H. Lago M.W. Rinehart J.K. Biller S.A. Gregg R.E. Wetterau J.R. Microsomal triglyceride transfer protein. Specificity of lipid binding and transport.J. Biol. Chem. 1995; 270: 6549-6554Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). This protein family also contains other members with a central role in animal reproduction and lipid circulation, such as vitellogenins, vertebrate's apoB, and insect apolipophorin (apoLp)-II/I (4.Babin P.J. Bogerd J. Kooiman F.P. Van Marrewijk W.J. Van der Horst D.J. Apolipophorin II/I, apolipoprotein B, vitellogenin, and microsomal triglyceride transfer protein genes are derived from a common ancestor.J. Mol. Evol. 1999; 49: 150-160Crossref PubMed Scopus (177) Google Scholar, 5.Wu L.T. Hui J.H. Chu K.H. Origin and evolution of yolk proteins: expansion and functional diversification of large lipid transfer protein superfamily.Biol. Reprod. 2013; 88: 102Crossref PubMed Scopus (20) Google Scholar). MTP is a heterodimeric protein complex composed of two distinct subunits, a large subunit of, typically, 99 kDa containing a lipid transfer activity and a multifunctional 58 kDa protein disulfide isomerase (PDI) (6.Wetterau J.R. Combs K.A. Spinner S.N. Joiner B.J. Protein disulfide isomerase is a component of the microsomal triglyceride transfer protein complex.J. Biol. Chem. 1990; 265: 9800-9807Abstract Full Text PDF PubMed Google Scholar, 7.Wetterau J.R. Zilversmit D.B. Purification and characterization of microsomal triglyceride and cholesteryl ester transfer protein from bovine liver microsomes.Chem. Phys. Lipids. 1985; 38: 205-222Crossref PubMed Scopus (100) Google Scholar). The MTP large subunit, for simplicity named only MTP, is composed of three structural motifs: the N-terminal β-barrel (N-sheet), the central α-helix domain and C-terminal β-sheet (C-sheet), and three functional domains (lipid transfer, membrane-associating, and apoB binding) (8.Hussain M.M. Shi J. Dreizen P. Microsomal triglyceride transfer protein and its role in apoB-lipoprotein assembly.J. Lipid Res. 2003; 44: 22-32Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar, 9.Mann C.J. Anderson T.A. Read J. Chester S.A. Harrison G.B. Kochl S. Ritchie P.J. Bradbury P. Hussain F.S. Amey J. et al.The structure of vitellogenin provides a molecular model for the assembly and secretion of atherogenic lipoproteins.J. Mol. Biol. 1999; 285: 391-408Crossref PubMed Scopus (170) Google Scholar). The N-sheet β-barrel is involved in the recognition of the N terminus of apoB, the central α-helix interacts with both apoB and PDI, and the C-sheet β-sheet has lipid binding as well as transfer properties (8.Hussain M.M. Shi J. Dreizen P. Microsomal triglyceride transfer protein and its role in apoB-lipoprotein assembly.J. Lipid Res. 2003; 44: 22-32Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar). In mammals, MTP is essential for the assembly and secretion of apoB-containing lipoprotein, chylomicrons in the intestine, and VLDLs in the liver (10.Raabe M. Veniant M.M. Sullivan M.A. Zlot C.H. Bjorkegren J. Nielsen L.B. Wong J.S. Hamilton R.L. Young S.G. Analysis of the role of microsomal triglyceride transfer protein in the liver of tissue-specific knockout mice.J. Clin. Invest. 1999; 103: 1287-1298Crossref PubMed Scopus (359) Google Scholar), and thereby facilitates delivery of triglyceride and cholesterol to the peripheral tissues. In humans, homozygous mutations in the MTP gene abolish secretion of apoB-lipoproteins and reduce the lipid level in plasma, resulting in abetalipoproteinemia (11.Wetterau J.R. Aggerbeck L.P. Bouma M.E. Eisenberg C. Munck A. Hermier M. Schmitz J. Gay G. Rader D.J. Gregg R.E. Absence of microsomal triglyceride transfer protein in individuals with abetalipoproteinemia.Science. 1992; 258: 999-1001Crossref PubMed Scopus (634) Google Scholar). Studies in the mice show that homozygous knockout of the MTP gene is lethal to the embryo (12.Raabe M. Flynn L.M. Zlot C.H. Wong J.S. Veniant M.M. Hamilton R.L. Young S.G. Knockout of the abetalipoproteinemia gene in mice: reduced lipoprotein secretion in heterozygotes and embryonic lethality in homozygotes.Proc. Natl. Acad. Sci. USA. 1998; 95: 8686-8691Crossref PubMed Scopus (220) Google Scholar). This phenotype is ascribed to the lack of lipoprotein synthesis and massive accumulation of lipid droplets in the cells of yolk sac endoderm (12.Raabe M. Flynn L.M. Zlot C.H. Wong J.S. Veniant M.M. Hamilton R.L. Young S.G. Knockout of the abetalipoproteinemia gene in mice: reduced lipoprotein secretion in heterozygotes and embryonic lethality in homozygotes.Proc. Natl. Acad. Sci. USA. 1998; 95: 8686-8691Crossref PubMed Scopus (220) Google Scholar), indicating that the yolk sac has lost its ability to produce lipoproteins and deliver lipids to the developing embryo. In oviparous organisms, such as frog (13.Sellers J.A. Hou L. Schoenberg D.R. Batistuzzo de Medeiros S.R. Wahli W. Shelness G.S. Microsomal triglyceride transfer protein promotes the secretion of Xenopus laevis vitellogenin A1.J. Biol. Chem. 2005; 280: 13902-13905Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), Drosophila (14.Palm W. Sampaio J.L. Brankatschk M. Carvalho M. Mahmoud A. Shevchenko A. Eaton S. Lipoproteins in Drosophila melanogaster–assembly, function, and influence on tissue lipid composition.PLoS Genet. 2012; 8: e1002828Crossref PubMed Scopus (149) Google Scholar, 15.Sellers J.A. Hou L. Athar H. Hussain M.M. Shelness G.S. A Drosophila microsomal triglyceride transfer protein homolog promotes the assembly and secretion of human apolipoprotein B. Implications for human and insect transport and metabolism.J. Biol. Chem. 2003; 278: 20367-20373Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), and worms (16.Shibata Y. Branicky R. Landaverde I.O. Hekimi S. Redox regulation of germline and vulval development in Caenorhabditis elegans.Science. 2003; 302: 1779-1782Crossref PubMed Scopus (99) Google Scholar), MTP has a similar function in the secretion of lipoproteins as found in mammals. The secretion of frog vitellogenin A-1 is MTP dependent (13.Sellers J.A. Hou L. Schoenberg D.R. Batistuzzo de Medeiros S.R. Wahli W. Shelness G.S. Microsomal triglyceride transfer protein promotes the secretion of Xenopus laevis vitellogenin A1.J. Biol. Chem. 2005; 280: 13902-13905Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), as the secretion is enhanced only when coexpressed with MTP. In Drosophila melanogaster, the transport of lipids between organs is carried out by a single apoB-family lipoprotein, lipophorin (Lpp) (14.Palm W. Sampaio J.L. Brankatschk M. Carvalho M. Mahmoud A. Shevchenko A. Eaton S. Lipoproteins in Drosophila melanogaster–assembly, function, and influence on tissue lipid composition.PLoS Genet. 2012; 8: e1002828Crossref PubMed Scopus (149) Google Scholar). The lipidation of Lpp occurs in two continuous steps with the help of two distinct lipid transfer proteins, MTP and large lipid transfer particle. Initially, Lpp is released from the fat body as a phospholipid-rich particle through the MTP-dependent mechanism and reaches the gut, where it is loaded with sterols and diacylglycerols via large lipid transfer particle. A homolog of the large subunit of MTP, named defecation suppressor of Clk (DSC-4), was found in the intestine of Caenorhabditis elegans (16.Shibata Y. Branicky R. Landaverde I.O. Hekimi S. Redox regulation of germline and vulval development in Caenorhabditis elegans.Science. 2003; 302: 1779-1782Crossref PubMed Scopus (99) Google Scholar). The salmon louse (Lepeophtheirus salmonis) is a marine ectoparasitic copepod that infests salmonids in the Northern Hemisphere. The salmon louse feeds on the blood, mucus, and skin of hosts and represents a major health and fish welfare issue that causes large economic losses in the Atlantic salmon (Salmo salar) farming industry (17.Westcott J.D. Hammell K.L. Burka J.F. Sea lice treatments, management practices and sea lice sampling methods on Atlantic salmon farms in the Bay of Fundy, New Brunswick, Canada.Aquacult. Res. 2004; 35: 784-792Crossref Scopus (46) Google Scholar) and also poses a considerable threat to wild salmonids (18.Costello M.J. How sea lice from salmon farms may cause wild salmonid declines in Europe and North America and be a threat to fishes elsewhere.Proc. Biol. Sci . 2009; 276: 3385-3394Crossref PubMed Scopus (223) Google Scholar). The lifecycle of the salmon louse consists, in total, of eight stages, each separated by a molt (19.Hamre L.A. Eichner C. Caipang C.M.A. Dalvin S.T. Bron J.E. Nilsen F. Boxshall G. Skern-Mauritzen R. The salmon louse Lepeophtheirus salmonis (Copepoda: Caligidae) life cycle has only two chalimus stages.PLoS One. 2013; 8: e73539Crossref PubMed Scopus (171) Google Scholar). The first are two stages of free-living nauplius larva followed by one infective copepodid stage. This is followed by two chalimus stages (where the parasites are firmly attached to the host), two preadult stages (with clear morphological sex difference), and finally the adult stage. Before host attachment, larvae are lecithotrophic, dependent on energy from maternally deposited lipid and protein reserves within the yolk (20.Tocher J.A. Dick J.R. Bron J.E. Shinn A.P. Tocher D.R. Lipid and fatty acid composition of parasitic caligid copepods belonging to the genus Lepeophtheirus.Comp. Biochem. Physiol. B. 2010; 156: 107-114Crossref PubMed Scopus (20) Google Scholar, 21.Dalvin S. Frost P. Loeffen P. Skern-Mauritzen R. Baban J. Ronnestad I. Nilsen F. Characterisation of two vitellogenins in the salmon louse Lepeophtheirus salmonis: molecular, functional and evolutional analysis.Dis. Aquat. Organ. 2011; 94: 211-224Crossref PubMed Scopus (39) Google Scholar). The sexually mature adult female continuously produces eggs carried in two egg-strings. Under laboratory conditions, female salmon lice can survive for at least 455 days and produce more than 11 pairs of egg-strings (22.Hamre L.A. Glover K.A. Nilsen F. Establishment and characterisation of salmon louse (Lepeophtheirus salmonis (Krøyer 1837)) laboratory strains.Parasitol. Int. 2009; 58: 451-460Crossref PubMed Scopus (108) Google Scholar). During egg production, the female louse incorporates massive amounts of yolk proteins (21.Dalvin S. Frost P. Loeffen P. Skern-Mauritzen R. Baban J. Ronnestad I. Nilsen F. Characterisation of two vitellogenins in the salmon louse Lepeophtheirus salmonis: molecular, functional and evolutional analysis.Dis. Aquat. Organ. 2011; 94: 211-224Crossref PubMed Scopus (39) Google Scholar, 23.Dalvin S. Frost P. Biering E. Hamre L.A. Eichner C. Krossoy B. Nilsen F. Functional characterisation of the maternal yolk-associated protein (LsYAP) utilising systemic RNA interference in the salmon louse (Lepeophtheirus salmonis) (Crustacea: Copepoda).Int. J. Parasitol. 2009; 39: 1407-1415Crossref PubMed Scopus (52) Google Scholar) and lipids into the growing oocytes. The predominant lipids in the eggs are neutral lipids, triacylglycerol, and cholesterol, followed by polar lipids, such as phosphatidylcholine and phosphatidylethanolamine, but fatty acid composition varies with the composition of the food received by the host salmon (20.Tocher J.A. Dick J.R. Bron J.E. Shinn A.P. Tocher D.R. Lipid and fatty acid composition of parasitic caligid copepods belonging to the genus Lepeophtheirus.Comp. Biochem. Physiol. B. 2010; 156: 107-114Crossref PubMed Scopus (20) Google Scholar). The mechanism of lipid accumulation in the growing oocytes has not been described in salmon lice. However, lipids are absorbed in the intestine and are likely to be transported with the hemolymph via lipoproteins, for example, to the oocytes. A highly efficient lipid uptake and transport can be predicted to secure the high production of eggs in salmon lice and dispersal of louse larvae in the environment. In the present study, we identified a gene encoding MTP from L. salmonis (LsMTP). We hypothesize that LsMTP may be involved in the lipoprotein-based supply of lipids from the intestine of a female salmon louse to growing oocytes. To this end, LsMTP was characterized and three transcript variants were identified. Silencing of the LsMTP gene using RNA interference (RNAi) affected production of eggs and reduced the viability of the developing larvae due to less neutral lipids in their yolk. Our results suggest that LsMTP has a crucial role in the reproduction of female salmon lice. A laboratory strain of salmon lice, L. salmonis (22.Hamre L.A. Glover K.A. Nilsen F. Establishment and characterisation of salmon louse (Lepeophtheirus salmonis (Krøyer 1837)) laboratory strains.Parasitol. Int. 2009; 58: 451-460Crossref PubMed Scopus (108) Google Scholar), was kept on Atlantic salmon (Salmo salar) in tanks with a continuous supply of seawater (temperature 10°C and salinity 34.5 ppt). Fish were fed a commercial diet daily. Nauplii I/II and copepodids were obtained from hatching egg-strings in hatching incubators supplied with the same seawater. Chalimus, preadult, and adult stages of lice were sampled from fish. Prior to sampling, fish were anesthetized with a mixture of benzocaine (60 mg/l) and metomidate (5 mg/l) in seawater. All the experiments and maintenance of salmon were carried out according to the Norwegian animal welfare legislation. For stage-specific quantitative (Q)-PCR, five biological replicates were collected from each stage. The following life stages and number of animals were collected for each replicate. Nauplius I (n = 100), nauplius II (n = 100), planktonic copepodid (n = 100), chalimus I (n = 10), chalimus II (n = 10), preadult I male and female (n = 1), preadult II male and female (n = 1), adult male (n = 1), young adult female and adult female (n = 1). For the starvation experiment, adult female lice were collected from fish and kept in seawater for 1–4 days. All the samples were stored in RNAlaterTM (Ambion) and kept overnight at 4°C prior to storing at −20°C for further use. Total RNA was extracted using TRI-reagent (Sigma-Aldrich) according to the manufacturer's instructions. The concentration and purity of isolated RNA was confirmed using Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies). The isolated total RNA samples were treated with amplification grade DNaseI (Invitrogen) as per manufacturer's instructions. For Q-PCR, DNase-treated total RNA (250 ng) was used for cDNA synthesis with Affinity Script QPCR cDNA synthesis kit (Stratagene) and diluted 10 times with nuclease-free water prior to storage at −20°C. For PCR, 1 μg total RNA was reverse transcribed using a qScript cDNA SuperMix (Quanta Bioscience). LsMTP-coding sequence was identified in the Ensembl database (http://r9ywwtvj.ensemblgenomes.org/Lepeophtheirus_salmonis/Info/Index) and the salmon louse genome database (accession: EMLSAT00000001530) (LiceBase, https://licebase.org/) with homology to known human (NCBI: X91148.1) and Drosophila MTP (FlyBase: FBgn0266369). The GenBank accession numbers of the three MTP sequences reported here are: LsMTP-A, MF063064; LsMTP-B, MF063065; and LsMTP-C, MF063066. PCR was carried out using GoTaq Flexi DNA polymerase (Promega) as per the manufacturer's protocol. The 5′ and 3′ rapid amplification of cDNA ends (RACE) was conducted with SMARTer RACE cDNA amplification kit (Clontech) as instructed in the users' manual. The 5′ and 3′ RACE-Ready cDNAs were synthesized from the total RNA of adult females and used for RACE-PCR. Gene-specific primers for 5′ and 3′ RACE are listed in supplemental Table S1. PCR products were cloned into pCR™ 4-TOPO® vector using the TOPO TA cloning kit for sequencing (Life Technologies) followed by transformation into Escherichia coli TOP10 cells. Clones were verified by PCR with M13 forward and reverse primers (supplemental Table S1). PCR products of positive clones were cleaned with ExoSAP-it (Affymetrix) and sequenced using BigDye Terminator v3.1 reagent (Applied Biosystems) at the sequencing facility of the University of Bergen. To confirm the in situ hybridization specificity, two different single stranded digoxigenin (DIG)-labeled RNA probes of 476 bp and 604 bp lengths corresponding to different regions of LsMTP transcripts (Fig. 1) were synthesized separately from cDNA using the DIG RNA labeling kit (Roche). Primers used for the synthesis of sense and antisense RNA probes are listed in supplemental Table S1. The concentration and labeling efficiency of probes was assessed by spectrometry (Nanodrop ND-1000) and with a spot test on nylon membrane, respectively. In situ hybridization was carried out in paraffin-embedded sections of adult female lice, as previously described by Kvamme, Frost, and Nilsen (24.Kvamme B.O. Frost P. Nilsen F. The cloning and characterisation of full-length trypsins from the salmon louse Lepeophtheirus salmonis.Mol. Biochem. Parasitol. 2004; 136: 303-307Crossref PubMed Scopus (18) Google Scholar) and Dalvin, Nilsen, and Skern-Mauritzen (25.Dalvin S. Nilsen F. Skern-Mauritzen R. Localization and transcription patterns of LsVasa, a molecular marker of germ cells in Lepeophtheirus salmonis (Kroyer).J. Nat. Hist. 2013; 47: 889-900Crossref Scopus (19) Google Scholar) with some modifications. Tissue sections were deparaffinized with Histoclear (National Diagnostic) instead of xylene and proteinase K treatment was done for 13 min. Hybridization of probes (500 ng/100 μl) was carried out at 65°C for 16–20 h. Sections were incubated with anti-DIG-alkaline phosphatase Fab fragments (Roche) and visualized using nitroblue tetrazolium (Roche) and 5-bromo-4-chloro-3-indolyl phosphate (Roche). The localization of LsMTP transcripts was detected with antisense probes and sense probes were used as negative controls. Q-PCR was performed on Applied Biosystem 7500 real-time PCR system using PowerUp SYBR Green Master Mix (Applied Biosystem) as per the manufacturer's recommendations. The Primers used in Q-PCR are listed in supplemental Table S1. The salmon louse elongation factor 1α (ef1α) was used as a reference (26.Frost P. Nilsen F. Validation of reference genes for transcription profiling in the salmon louse, Lepeophtheirus salmonis, by quantitative real-time PCR.Vet. Parasitol. 2003; 118: 169-174Crossref PubMed Scopus (103) Google Scholar). Two-fold serial dilutions (six dilutions) of cDNA were used to create a standard curve for efficiency calculation. As the efficiency of the assay ranged from 95% to 100%, all the assays were carried out simultaneously for LsMTP and ef1α using the same cDNA and master mix along with two negative controls, a nontemplate control and a no reverse transcriptase control. All the samples were run in duplicate, and Ct (cycle threshold) values were averaged. The final results were analyzed using the 2−ΔΔCT method (27.Kvamme B.O. Skern R. Frost P. Nilsen F. Molecular characterisation of five trypsin-like peptidase transcripts from the salmon louse (Lepeophtheirus salmonis) intestine.Int. J. Parasitol. 2004; 34: 823-832Crossref PubMed Scopus (43) Google Scholar). The Q-PCR analysis was performed on lice recovered from two RNAi experiments. Primers used in Q-PCR for the detection of downregulation in the RNAi experiments were designed outside the double-stranded RNA (dsRNA) fragments. For each RNAi experiment, five representative adult females from the control group and LsMTP dsRNA-treated group were analyzed. Animals from the control group were used as a calibrator to calculate relative expression. Relative expression levels of three variants of LsMTP in different developmental stages of salmon lice were also determined by Q-PCR using copepodids as a calibrator. For the starvation experiment, animals (n = 5) were collected on days 0, 1, 2, and 4, and after 2 days of refeeding on the host fish. Relative expression of LsMTP was calculated using day 4 for calibration. dsRNA was prepared according to the Megascript RNAi kit (Ambion). Two different fragments targeting different regions of LsMTP mRNA (Fig. 1) were amplified by PCR from primers with T7 promoter sequence, previously used for synthesis of in situ hybridization probes (supplemental Table S1). A fragment of 850 bp from cod trypsin (CPY185) was used as a control (23.Dalvin S. Frost P. Biering E. Hamre L.A. Eichner C. Krossoy B. Nilsen F. Functional characterisation of the maternal yolk-associated protein (LsYAP) utilising systemic RNA interference in the salmon louse (Lepeophtheirus salmonis) (Crustacea: Copepoda).Int. J. Parasitol. 2009; 39: 1407-1415Crossref PubMed Scopus (52) Google Scholar). Respective PCR products were used as templates for the synthesis of sense and antisense RNAs by in vitro transcription using T7 polymerase. For synthesis of dsRNA sense and antisense, RNAs were pooled and incubated at 75°C for 5 min followed by slow cooling to room temperature. The purified dsRNA concentrations were measured with Nanodrop ND 1000 Spectrophotometer, and a final concentration of 600 ng/μl was used for injections. Two RNAi experiments were conducted separately in female lice. The first knockdown of LsMTP was carried out with dsRNA fragment 1 (LsMTP Fr 1) in newly molted preadult II females. In the second experiment, LsMTP silencing was done with dsRNA fragment 2 (LsMTP Fr 2) in young adult female lice. Both RNAi experiments were performed as described by Dalvin et al. (23.Dalvin S. Frost P. Biering E. Hamre L.A. Eichner C. Krossoy B. Nilsen F. Functional characterisation of the maternal yolk-associated protein (LsYAP) utilising systemic RNA interference in the salmon louse (Lepeophtheirus salmonis) (Crustacea: Copepoda).Int. J. Parasitol. 2009; 39: 1407-1415Crossref PubMed Scopus (52) Google Scholar). In each experiment, female lice were injected for LsMTP dsRNA and control dsRNA separately. After injection of dsRNA, lice were kept in seawater for 3 h and put back (n = 30–32) on three fish for every dsRNA fragment with equal numbers of dsRNA-treated female and untreated male lice. Both RNAi experiments were terminated when control dsRNA-injected female lice produced second pairs of egg-strings. Female lice were examined for gross morphology and imaged along with egg-strings for further egg-string measurement. Afterwards, egg-strings from females of both experiments were removed gently with forceps, placed into individual hatching incubators and closely examined every day. The offspring from the first RNAi experiment were evaluated visually and counted at 9 days post hatching when control animals had developed to copepodids. Nauplii from the second RNAi experiment were collected, visually evaluated, and counted between 6 and 8 h post hatching and closely followed through molting to nauplii II and further to copepodids. Neutral lipid content was detected and quantified in nauplii I using lipid stains (see below). Seven independent replicates of groups of 25 nauplii hatched from control and LsMTP dsRNA-injected female egg-strings from the second RNAi experiment were used for qualitative and semi-quantitative analysis of neutral lipids. Nauplii I were collected from hatching incubators, washed three times with cold PBS, and fixed in phosphate-buffered 4% paraformaldehyde (pH 7.4) for 2 h. Oil Red O stain was performed using the methods described (28.Nishiura J.T. Burgos C. Aya S. Goryacheva Y. Lo W.Y. Modulation of larval nutrition affects midgut neutral lipid storage and temporal pattern of transcription factor expression during mosquito metamorphosis.J. Insect Physiol. 2007; 53: 47-58Crossref PubMed Scopus (24) Google Scholar) with some modifications. Fixed nauplii were washed three times with cold PBS, resuspended in 60% isopropanol for 10 min, and stained with Oil Red O stain (Sigma-Aldrich) for 30 min. After staining, nauplii were washed in cold PBS, rinsed with 60% isopropanol, mounted, and photographed with a Leica Model MZ6 stereo microscope. Nile Red stain was used to detect neutral lipids in unfixed nauplii I according to (29.Baer M.M. Palm W. Eaton S. Leptin M. Affolter M. Microsomal triacylglycerol transfer protein (MTP) is required to expand tracheal lumen in Drosophila in a cell-autonomous manner.J. Cell Sci. 2012; 125: 6038-6048Crossref PubMed Scopus (13) Google Scholar) with the following modifications. Nauplii I were washed three times with cold PBS, stained with 1 ug/ml of Nile Red (Sigma-Aldrich) in PBS for 30 min, and imaged directly with a Leica TCS SP5 confocal microscope. Neutral lipids were visualized by excitation at 543 nm and fluorescence detection at 635 nm. The semi-quantification of total neutral lipids of nauplii I was carried out using Oil Red O stain. After fixation and staining with Oil Red O, the excess stain was washed away with 60% isopropanol. Oil Red O stain was extracted from nauplii using 200 μl of 100% isopropanol and absorbance was measured at 500 nm in duplicate. Background signal was subtracted using 100% isopropanol as a background control. The Staden package (30.Staden R. Beal K.F. Bonfield J.K. The Staden package, 1998.Methods Mol. Biol. 2000; 132: 115-130PubMed Google Scholar) was used for DNA sequence assembly, editing, and analysis. Multiple sequence alignment was done in BioEdit version 7.2.5 (31.Hall T. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT.Nucleic Acids Symp. Ser. 1999; 41: 95-98Google Scholar) using ClustalW. Accession numbers of MTP protein sequences from other species included in the multiple alignment were as follows: Homo sapiens (NCBI: NP_000244.2), Salmo salar (NCBI: XP_014050992.1), Danio rerio (NCBI: NP_998135.1), D. melanogaster (NCBI: NP_610075.2), Zootermopsis nevadensis (NCBI: KDR21635.1), Daphnia magna (NCBI: JAN30039.1), Scylla olivacea (Uniport: A0A0P4WDH4), and Xenopus tropicalis (NCBI: XP_002934813.1). Signal peptides were predicted using Phobius (http://phobius.sbc.su.se/) and SignalP server (http://www.cbs.dtu.dk/services/SignalP/). Conserved domain (LpD-N) in the protein sequences was analyzed in Conserved Domain Database (32.Marchler-Bauer A. Derbyshire M.K. Gonzales N.R. Lu S. Chitsaz F. Geer L.Y. Geer R.C. He J. Gwadz M. Hurwitz D.I. et al.CDD: NCBI's conserved domain database.Nucleic Acids Res. 2015; 43: D222-D226Crossref PubMed Scopus (2256) Google Scholar). Secondary structures of proteins were predicted using JPred4 (33.Drozdetskiy A. Cole C. Procter J. Barton G.J. JPred4: a protein secondary structure prediction server.Nucleic Acids Res. 2015; 43: W389-W394Crossref PubMed Scopus (1095) Google Scholar) or PSSpred (http://zhanglab.ccmb.med.umich.edu/PSSpred/) and three-dimensiona" @default.
• W2621590622 created "2017-06-15" @default.
• W2621590622 creator A5033002724 @default.
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• W2621590622 creator A5075646749 @default.
• W2621590622 creator A5090512788 @default.
• W2621590622 date "2017-08-01" @default.
• W2621590622 modified "2023-10-04" @default.
• W2621590622 title "Microsomal triglyceride transfer protein in the ectoparasitic crustacean salmon louse (Lepeophtheirus salmonis)" @default.
• W2621590622 cites W1495890492 @default.
• W2621590622 cites W1563885314 @default.
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