Investigation of Hox Gene Expression in the Brazilian Whiteknee Tarantula Acanthoscurria Geniculata

Investigation of Hox gene expression in the Brazilian Whiteknee tarantula Acanthoscurria geniculata Dan Strömbäck Degree project in biology, Bachelor of science, 2020 Examensarbete i biologi 15 hp till kandidatexamen, 2020 Biology Education Centre and Institutionen för biologisk grundutbildning vid Uppsala universitet, Uppsala University Supervisor: Ralf Janssen Abstract Acanthoscurria geniculata, the Brazilian whiteknee tarantula, is part of the group Mygalomorphae (mygalomorph spiders). Mygalomorphae and Araneomorphae (true spiders) and Mesothelae (segmented spiders) make up Araneae (all spiders). All spiders have a prosoma with a pair of chelicerae, pedipalps and four pairs of legs, and an opisthosoma with two pairs of book lungs or one pair of book lungs and one pair of trachea (in opisthosomal segments O2 and O3) and one or two pairs of spinnerets (in segments O4 and O5). The mygalomorphs have retained two pairs of book lungs, an ancestral trait evident from looking at Mesothelae, the ancestral sister group of both Araneomorphae and Mygalomorphae. The spinnerets differ greatly between the groups, but this study focuses on comparing Mygalomorphae and Araneomorphae. Mygalomorphae also have reduced anterior spinnerets, but instead enormous posterior spinnerets. Araneomorphae possess all four, but none particularly big. The genetic basis of these differences between the set of opisthosomal appendages in tarantulas and true spiders is unclear. One group of genes that could be involved in the development of these differences could be the famous Hox genes. Hox genes have homeotic functions. If they are expressed differently between these two groups, the resulting morphology could change. This study focuses on the posterior Hox genes in A. geniculata, i.e. Antennapedia, Ultrabithorax, AbdominalA, and AbdominalB, each present in the form of two paralogs. Differences found in the expression patterns among these three spiders can explain structural differences in the opisthosoma. Introduction The Brazilian whiteknee tarantula, Acanthoscurria geniculata, belongs to the infraorder Mygalomorphae, a sister group of Araneomorphae (true spiders). They, along with the ancestral sister group Mesothelae (segmented spiders), make up the order Araneae. All spiders have some unique physical traits in common, such as the spinnerets, which are organs used to spin silk. There are morphological differences between Mygalomorphae and Araneomorphae. Mygalomorphae are often much larger, and their chelicerates are angled differently from those in Araneomorphae. Mygalomorphae can only move their fangs vertically, whereas the fangs in Araneomorphae are angled toward each other. Mygalomorphae have also retained two pairs of book lungs, whereas in true spiders the posterior pair has been reduced to tracheae (Wheeler et al., 2016). Two pairs of book lungs are likely ancestral as the ancestral group Mesothelae also has two pairs (Küntzel et al., 2019). Other differences concern the spinnerets. Normally there are two pairs in the opisthosoma, two anterior in segment O4 and two posterior in segment O5. In Mygalomorphae, the anterior pair is reduced, but the posterior spinnerets are very large compared to those of Araneomorphae (Mariano-Martins et al., 2020; Wheeler et al., 2016). These morphological differences could be explained by the function of the famous Hox genes. Hox genes are genetic “switches'' mainly active in the embryonic stage that turn on or off downstream cascades that determine the identity of most segments (such as leg- , chelicerate- or spinneret-bearing segments). Their location on the chromosomes is in clusters, meaning that they are all next to one another (Pace et al., 2016). Interestingly, they are expressed in order of occurrence on the chromosome, and this is called co-linearity. The first Hox gene is activated earliest and is expressed most anteriorly in the embryo, the other Hox genes are then expressed one after one more and more posteriorly (Barnett & Thomas, 2013; Khadjeh et al., 2012; Schwager et al., 2017). There are ten Hox genes in all arthropods (Schwager et al., 2017). However, in some groups, like spiders, often two paralogs exist, as a result of a whole genome duplication (Schwager et al., 2017). The expression of these genes has previously been studied in true spiders, among them the common house spider Parasteatoda tepidariorum and the Central American wandering spider Cupiennius salei (e.g. Damen, 1998; Khadjeh et al., 2012; Schwager et al. (2007, (2017))), but although the genes have been identified in A. geniculata (Janssen, unpublished data), and relationship between the genes in A. geniculata and P. tepidariorum has been determined with a phylogenetic analysis (Figure 1), and two of these genes, Antp-2 and AbdA-2 have been cloned (Janssen, unpublished data), there are no expression patterns of any tarantula Hox gene thus far. Presence and relationship between the genes in A. geniculata and P. tepidariorum has been determined with a genetic analysis (Figure 1), and two of these genes, Antp-2 and AbdA-2 have been cloned, but there are no expression patterns of any tarantula Hox gene thus far. This study focuses on the comparison of the four most posterior Hox genes, each present in the form of two paralogs, Antennapedia-1 (Ag-Antp-1) and Antennapedia-2 (Ag-Antp-2), Ultrabithorax-1 (Ag-Ubx-1) and Ultrabithorax-2 (Ag-Ubx-2), AbdominalA-1 (Ag-AbdA-1) and AbdominalA-2 (Ag-AbdA-2), AbdominalB-1 (Ag-AbdB-1) and AbdominalB-2 (Ag-AbdB-2). This is because these genes are involved in the formation of the opisthosomal structures book lungs (as well as tracheae in true spiders) and spinnerets (e.g. Schwager et al. 2017). Figure 1: Phylogenetic tree of Hox genes, Antennapedia, Ultrabithorax, AbdominalA, and AbdominalB based on entire open reading frames (Janssen, unpublished data) to show the phylogenetic relationship between the genes in Parasteatoda tepidariorum and Acanthoscurria geniculata. It suggests that each posterior Hox gene in Pt possesses one ortholog in Ag. Methods Gene cloning The genes worked on from the start were Ag-Ubx-1, Ag-Ubx-2, Ag-AbdA-1, Ag-Antp-1 and Ag- AbdB-1. The others, Ag-AbdA-2, and Ag-Antp-2, were isolated before (Janssen, unpublished data). Primers (see Appendix A) for each gene were obtained from Macrogen and diluted to a 100mM solution. 1 µl of each primer (forward and backward primers) were added to separate tubes, along with 14.8 µl H2O, 2.5 µl 10x buffer solution, 2.5 µl of 2 mM dNTPs, 1.5 µl 25 mM MgCl2, 1 µl cDNA and 0.2 µl 5 U/µl Taq-polymerase. After the first PCR, a second PCR was done with the same ingredients, but with an inner pair of primers and 1 µl of the first PCR product instead of cDNA. DNA ligation and bacterial transformation Two methods were used to ligate fragments into bacterial vectors. The first, used on Ag-AbdA- 1 and Ag-Ubx-2, was a topoisomerase ligation reaction. 3 µl of the PCR product, 1 µl H2O, 1 µl salt solution and 1 µl TOPO vector (ThermoFisher) were added to a tube and incubated at room temperature for 15 minutes. 2 µl of this mix was added to a tube of Escherichia coli on ice and incubated for 20 minutes. The second method was ligation with a T4 ligase, this was done on Ag-AbdB-1 and Ag-Antp- 1. 2.5 µl of each gene from second PCR product was added to a tube each with 1 µl 5x buffer solution, 1 µl vector and 0.5 µl T4-DNA ligase. 2 µl were added to tubes with chemically competent cells on ice and incubated for 20 minutes. Bacteria plus vectors were heat-shocked in a water bath at 42°C for 30 seconds, after which 250 µl of SOC-medium was added immediately after the tubes were briefly put into ice. This was done regardless of the previous ligation method. They were then incubated in a shaker at 37°C for one hour. The contents were added tospread on agar plates of with kanamycin treated with and 50µl X-gal in dimethylformamide and incubated overnight at 37°C. Kanamycin allows only bacteria that have been transformated (taken up a vector) to grow, because the kanamycin-resistance gene is located on the vector. X-gal allows for the distinguishing of self- ligated vectors from vectors with an insert (PCR fragment). Blue colonies are self-ligated, white colonies represent colonies with an insert; insertion of a PCR fragment destroys the beta- Gal gene located at the insertion site on the vector. This beta-Gal gene would be needed to metabolize the X-gal (the metabolic product is blue). Colony PCR A master mix for each PCR was made, containing 64 µl H2O, 10 µl 10x buffer, 10 µl of 2mM dNTPs, 8 µl 25 mM MgCl2, 4 µl of 20 mM M13 forward primer, 4 µl of 20 mM M13 reverse primer and 0.5 µl 5 U/µl Taq-polymerase. This was then divided into eight PCR tubes (with 10 µl each of the master mix) per gene. Eight white colonies per plate were chosen and placed in a PCR tube each (and then dabbed on a control plate). A gel electrophoresis was run to determine whether the bacteria had picked up a fragment corresponding to the right size, and a maximum of two of those were chosen for subsequent sequencing. Chosen colonies were picked from the control plate with a pipette tip and placed into a tube (half-open lid for oxygen access) with 3 ml LB-medium + kanamycin. These were then incubated overnight in a shaker at 37℃. Plasmid extraction To extract plasmids from the selected bacteria, a ThermoFisher Plasmid DNA miniprep kit was used following the instructions provided, with the following changes; the neutralisation solution was added after three minutes instead of five, and 80 µl elution solution was used instead of 100 µl.

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