Alves, Carla et al. (2021) International Worm Meeting "The effects of venlafaxine on behavior and central nervous system of Caenorhabditis elegans"

Vanin, Ana Paula, dos Santos, Amanda, Sutorillo, Nathalia Tafarel, Barcellos, Leonardo Jose Gill, Santini, Wallace, Alves, Carla, Tamagno, Wagner Antonio, Kaizer Perin, Rosilene Rodrigues

[International Worm Meeting, 2021]

The growing consumption of psychoactive drugs, including antidepressants and antipsychotics, along with the low levels of total metabolization of these drugs, are increasing their residues in the environment. After being used, the drugs are metabolized and excreted, sometimes still in their active form, and may or may not suffer degradation when in contact with the environment. Thus, these psychoactive drugs can reach the aquatic environment and affect non-target organisms, such as zebrafish (Danio rerio) and Caenorhabditis elegans, in their natural environments. Our main hypothesis is that venlafaxine (VEN), a drug that works with the selective inhibition of the reuptake of noradrenaline and serotonin, can exert effects on the endocrine and behavioral system of zebrafish and toxicological effects on the behavior and nervous system of C. elegans. The integrity of the natural behavioral repertoire is vital for the maintenance of the species, reinforcing the importance of studies that show the potential of drugs in altering these models. Here we evaluate acute exposure to VEN, in three concentrations followed by a behavioral assessment. The behavior of C. elegans was analyzed by the body bands, pharyngeal beat, and defecation cycle; while the biochemical analyzes of the enzyme Acetylcholinesterase to assess the toxicological effects of VEN on the nervous system. We have shown that the nematode C. elegans resists changes in behavior and the nervous system. Thus, this model response was not a clear indicator of water contamination and possible zebrafish effects, probably because of the cuticle and natural behavior of the nematode. Results with zebrafish exposure to VEN have shown changes in the novel tank test, mainly the robust anxiolytic-like pattern, suggesting that VEN may be more dangerous to species conservation, since fish presenting an anxiolytic-like behavior becomes more susceptible to predation. We are working on biomarkers to study differences in the responses to these models when exposed at VEN.

Tamagno, Wagner Antoino et al. (2021) International Worm Meeting "Protective and reparative effect of dragon fruit upon central nervous system toxicity induced by copper"

Tamagno, Wagner Antoino, Sutorillo, Nathalia Tafarel, Alves, Carla, dos Santos, Amanda, Kaizer, Rosilene Rodrigues, Barcellos, Leonardo Jose Gill, Santini, Wallace, Vanin, Ana Paula

[International Worm Meeting, 2021]

Copper is an essential metal and is important in general metabolism. However, in high concentrations, it becomes toxic to the organism. The metal-induced toxicity is linked to many neurodegenerative diseases like Parkinson's, Alzheimer's, multiple sclerosis, occurring in senile aging. To prevent neurodegenerative diseases, some studies are being developed to find ways of healthy aging. Natural compounds and diets based on fruits are increasing. Dragon fruit (Hylocereus undatus) is a tropical and Latin American fruit that is gaining more popularity due to its antioxidant properties. This fruit still has low popularity, since its planting and large-scale commercialization are recent. Here we evaluate the protective and reparative effect of different doses of dragon fruit's microencapsulated pulp extract on copper-induced toxicity. The nematode Caenorhabditis elegans was used as a model, to research the effects of pitaya extract on the Cholinergic nervous system, behavior, lipidic peroxidation, and chaperon system. We observed that Cu severally increased directly the cholinesterase, chaperone, and peroxidation rates. When pitaya is applied, even to prevent or remediate, all those enzymes' rates are normalized again. Overall, the results have shown that the pulp fruit extract can be used on dietary supplementation to prevent and repair neural damage. The extract was able to regulates the chaperone system to reduce expression of heat shock protein (16.2) and reestablished the AChE levels avoiding lipidic peroxidation. Changes in the behavior, decreasing cell death biomarkers, and lipidic peroxidation caused by copper toxicity are showing, and, based on these results, we concluded that the pitaya fruit has an important role in gerontological issues.

Giordano-Santini, R. et al. (2011) International Worm Meeting "MosSCI-biotic: neomycin resistance gene as a co-insertion marker for Mos1-mediated single-copy insertion (MosSCI)."

Giordano-Santini, R., Johnsen, R., Dupuy, D., Baillie, D., Tu, D.

[International Worm Meeting, 2011]

Since the development of Mos1-mediated single-copy insertion (MosSCI) a few years ago, MosSCI has become the method of choice to introduce a single-copy of transgenes in the C. elegans genome (Frokjaer-Jensen, C. et al., 2008; Nat. Genet. 40(11):1375-1383). This transposon-excision repair method has been developed using unc-119 as a co-insertion marker, and two intergenic Mos1 insertion sites have been used to date. In theory, every Mos1 intergenic insertion from NemaGENETAG could be used for gene conversion and single-copy integration using MosSCI. However, the use of unc-119 as a co-insertion marker necessitates the introduction of the unc-119(ed3) allele in the genetic background beforehand, a requirement that may not be suitable for cases where the unc-119(ed3) mutant background may interfere with the study or if the desired Mos1 insertion site is too close to the unc-119 locus. We have previously shown that the neomycin resistance gene (neoR) is a potent genetic marker for nematode transgenesis and that a single-copy of neoR is enough to confer resistance to G-418 in wild type animals (Giordano-Santini, R. et al., 2010; Nat. Methods 7(9):721-723). By using two different Mos1 insertion lines, we demonstrated that this powerful selection system could be used in the context of MosSCI and replace the unc-119 co-insertion marker. Herein, we show that neoR is compatible with the MosSCI heat-shock protocol as well as the direct protocol, in which the Transposase source is under the control of a heat-shock promoter or under the promoter of a germline specific gene respectively. We demonstrate that the majority of steps in these protocols are facilitated by the use of antibiotic resistance systems, hence proving that the benefits of antibiotic selection extend to single-copy transgene applications. We therefore believe that these benefits should contribute to the development of more flexible and efficient techniques for nematode transgenesis.

Frokjaer-Jensen, Christian et al. (2011) International Worm Meeting "A recombinant Mos1 transposon can carry large DNA fragments."

Stevenson, ZC, Sarov, M, Taylor, J, Frokjaer-Jensen, Christian, Davis, MW, Moerman, D, Jorgensen, EM

[International Worm Meeting, 2011]

P element-mediated transgenesis has been an important tool in Drosophila for the past 30 years. For example, P-elements have been used extensively to insert transgenes, to generate loss of function mutants and to probe the genome for regulatory regions. We have developed a minimal recombinant Mos1A transposon (miniMos) that behaves much like a P-element. Exogenous DNA placed inside a recombinant transposon is inserted into the C. elegans genome at high frequencies by simple injection (60% insertion freq.) or by heat-shock. In characterized lines, 46% inserted in intergenic regions, 43% in introns and 11% in exons (n = 54). The method is easy, very robust and works well with different mutant selection markers (unc-119 and unc-18) as well as antibiotic markers (neomycinB and puromycinC). At lower frequencies (2-14%), miniMos can carry large pieces of DNA derived from fosmids (45kb). We have verified by comparative genome hybridization (CGH) that fully intact fosmids are inserted and all 18 independent air-2::gfp fosmid insertions express visible GFP. This technique makes the generation of single-copy transgenes rapid and easy. In addition, miniMos transgenes can be used for gene-traps and for genome-wide chromatin studies. Gene trapping. The miniMos element can be used to simultaneously disrupt genes and to determine their expression pattern. We have constructed a miniMos gene-trap carrying a promoterless mCherry with an SL2 trans-splice leader sequence. In a pilot screen, we have isolated 20 lines that express mCherry in specific patterns. Each line contains a transposon inserted into a transcription unit (either exon, intron or UTR). Chromatin and gene regulation. The miniMos element can be used to probe the genome for permissive and silenced regions. Germline expressed genes are highly enriched in introns that contain phased DNAD and are virtually absent from the X chromosomeE. We can test the effects of genomic environment and phasing by inserting germline expressed transgenes. For example, a phased Ppie-1::GFP::H2B construct results in stable germline fluorescence in 67% (12 of 18) of inserts whereas similar, but non-phased Pgld-1 and Pmex-5 constructs result in much lower frequencies of fluorescence. (A) Bessereau et al., (2001) (B) Giordano-Santini et al., (2010) (C) Semple et al., (2010) (D) Fire et al., (2006) (E) Bender et al., (2006) .

Jon Pierce-Shimomura et al. (2004) West Coast Worm Meeting "Swimming as a distinct form of locomotion in C. elegans: Analysis of the switch between crawling and swimming"

Steve McIntire, Jon Pierce-Shimomura

[West Coast Worm Meeting, 2004]

C. elegans crawls on a flat surface and swims in liquid. Both forms of locomotion are initiated as a series of alternating dorsal-ventral head bends. The DV bends propagate backward along the body to generate forward thrust. The two behaviors are distinct in that swimming bends are deeper and more rapid than crawling bends (Crawl ~0.5 Hz, Swim ~2.0 Hz). These differences underlie the characteristic body waveforms for each behavior. When the head is bent maximally to the ventral or dorsal side, the body forms a 'C' shape when swimming, and an 'S' shape when crawling. The motion of C. elegans in liquid has previously been called 'thrashing,' implying that the worm flails its body in an uncontrolled fashion. We find, however, that C. elegans makes efficient progress when swimming over a surface to orient up an attractive salt gradient. Therefore, for C. elegans , as for other nematode species (Wallace, 1958 Ann Applied Bio ), movement through liquid can be a goal-oriented form of locomotion that may be better described as 'swimming'. Swimming may have important adaptive value for C. elegans in its natural environment of damp soil where it must traverse through pockets of water. C. elegans rapidly switches between crawl and swim kinematics when it enters and exits a puddle. This switch is reminiscent of how other animals switch between distinct forms of locomotion called gaits. In some animals, gaits are known to be controlled by neural networks that are distinct in either the sense that separate networks control each gait, or a single locomotion network operates in different 'gait' states. We wondered whether distinct neural networks may also control crawling and swimming in C. elegans . To better understand swimming behavior and the switch from crawling to swimming, we screened for mutants that are normal for crawling, but abnormal for the initiation or maintenance of swimming, what we term 'Swa' ( Sw imming a bnormal). We isolated over 40 swa mutants and divided them into different classes. Interestingly, all of these mutants exhibit normal crawling. Four Swa classes related to the maintenance of swimming included those with smaller amplitude of head bends, irregular swim pattern, extra left-right bends, and frequent stopping. Two Swa classes related to the initiation and maintenance of swimming included (1) those that in liquid exhibit persistent crawling, and (2) those that exhibit delayed initiation together with frequent stopping. This second class included several mutants with defective touch responses suggesting importance of mechanosensory input in swimming. Analysis of mechanosensory mutants revealed that specific loss of function of the six main mechanosensory neurons (e.g. mec-3 and mec-4 ) results in only a mild irregular swim pattern. Mechanosensory mutants with defective mechanosensory and chemosensory function ( mec-1 , mec-2 and mec-8 ), however, exhibit delayed swim initiation, severely irregular swim pattern, and frequent stopping. Analysis of chemosensory mutants revealed that loss of function in taste (e.g. che-1 ), but not olfaction (e.g. odr-1 odr-7 ), produced a similar severe swim phenotype as observed in mec-2 mutants. Chemosensory input may be particularly important for the initiation and maintenance of swimming. Our findings demonstrate that crawling and swimming are distinct forms of locomotion for C. elegans . Moreover, both mechanosensory and chemosensory pathways appear to control the initiation and maintenance of swimming. Analysis of the switch between crawling and swimming in C. elegans may help to uncover the general neurogenetic mechanisms that underlie the control of patterned neural output.