Daniel Fenando Paulo- PhD
- Junior Researcher at University of Hawaiʻi at Mānoa
Daniel Fenando Paulo
- PhD
- Junior Researcher at University of Hawaiʻi at Mānoa
genetics, evolution, and functional genomics of tropical insect pests
About
16
Publications
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Introduction
I am an enthusiastic scientist and researcher inspired by biology, broadly interested in the evolutionary origins and genetic basis of ecological adaptations and speciation in insects, particularly pest species. During my academic career, I have acquired vast experience in the development of scientific and technology research projects, as well as in genomics and transcriptomics analysis, methods in molecular biology, and genome editing (CRISPR/Cas9) in the field of animal genetics and evolution.
Current institution
Additional affiliations
June 2008 - May 2021
Publications
Publications (16)
For almost a decade, natural populations of the South American fruit fly have been targeted for control through Sterile Insect Technique projects. To ensure a sustainable supply of competitive sterile flies for this approach, it is essential to understand the effects of domestication when strains of this pest are initially brought into the laborato...
The remarkable diversity of insect pigmentation offers a captivating avenue for studying evolution and genetics. In tephritids, understanding the molecular basis of mutant traits is also crucial for applied entomology, enabling the creation of genetic sexing strains through genome editing, thus facilitating sex-sorting before sterile insect release...
The Mexican fruit fly, Anastrepha ludens, is a polyphagous true fruit fly (Diptera: Tephritidae) considered one of the most serious insect pests in Central and North America to various economically relevant fruits. Despite its agricultural relevance, a high-quality genome assembly has not been reported. Here, we described the generation of a chromo...
Tephritid fruit flies are among the most invasive and destructive agricultural pests worldwide. Over recent years, many studies have implemented the CRISPR/Cas9 genome-editing technology to dissect gene functions in tephritids and create new strains to facilitate their genetics, management, and control. This growing literature allows us to compare...
Blowflies are of interest for medical applications (maggot therapy), forensic investigations, and for evolutionary developmental studies such as the evolution of parasitism. It is because of the latter that some blowflies such as the New World screwworm and the Australian sheep blowfly are considered major economic pests of livestock. Due to their...
The evolution of obligate ectoparasitism in blowflies (Diptera: Calliphoridae) has intrigued scientists for over a century, and surprisingly, the genetics underlying this lifestyle remain largely unknown. Blowflies use odors to locate food and oviposition sites; therefore, olfaction might have played a central role in niche specialization within th...
To what extent can we predict how evolution occurs? Do genetic architectures and developmental processes canalize the evolution of similar outcomes in a predictable manner? Or do historical contingencies impose alternative pathways to answer the same challenge? Examples of Müllerian mimicry between distantly related butterfly species provide natura...
Cochliomyia hominivorax and Lucilia cuprina are major pests of livestock. Their larvae infest warm-blooded vertebrates and feed on host’s tissues, resulting in severe industry losses. As they are serious pests, considerable effort has been made to develop genomic resources and functional tools aiming to improve their management and control. Here, w...
In 2013, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) was officially declared as present in Brazil and, after two years, the species was detected in the Caribbean and North America. Information on genetic features and accurate distribution of pests is the basis for agricultural protection policies. Furthermore, such knowledge is imperativ...
Blowflies and houseflies are mechanical vectors inhabiting synanthropic environments around the world. They feed and breed in fecal and decaying organic matter, but the microbiome they harbour and transport is largely uncharacterized. We sampled 116 individual houseflies and blowflies from varying habitats on three continents and subjected them to...
MicroRNAs (miRNAs) are small noncoding RNAs that modulate gene expression through post-transcriptional regulation. Here, we report the identification and characterization of miRNAs in two closely related screwworm flies with different feeding habits: Cochliomyia hominivorax and Cochliomyia macellaria. The New World screwworm, C. hominivorax, is an...
True flies are insects of the order Diptera and encompass one of the most diverse groups of animals on
Earth. Within dipterans, Schizophora represents a recent radiation of insects that was used as a model
to develop a pipeline for generating complete mitogenomes using various sequencing platforms and
strategies. 91 mitogenomes from 32 different sp...
Species identification is an essential step in the progress and completion of work in several areas of biological knowledge, but it is not a simple process. Due to the close phylogenetic relationship of certain species, morphological characters are not always sufficiently distinguishable. As a result, it is necessary to combine several methods of a...
The cotton bollworm, Helicoverpa armigera (Hübner), was recently introduced in Brazil. During the 2012‐2013 harvest, producers reported reduced yields up to 35% on major crops. The economic losses reached US$ 1 billion only in western Bahia, triggering a phytosanitary crisis. The deficiencies in existing taxonomic keys to deal with the morphologica...
MicroRNAs (miRNAs) are small non-coding RNAs that act as post-transcriptional modulators of gene expression in Eukaryotes. The imperfect complementarity between miRNAs and the 3’ untranslated region of mRNAs inhibits their translation in animals, being key genes for control of expression in cells. The characterization of miRNAs provides a better un...
The superfamily Oestroidea, comprising ∼15,000 species, is a large and ecologically diverse clade within the order Diptera. Among its six commonly recognized families, Calliphoridae seems to be crucial for understanding evolutionary relationships in the group, as it is recognized as a controversial paraphyletic grouping. To further investigate this...
Questions
Questions (9)
Hi all,
I made a double digestion of a plasmid (1 ug) using 20U of each EagI-HF and NcoI-HF REs and 1x rCutSmart buffer in a 50 ul final volume. The reaction was incubated at 37 ˚C overnight. On the next day, I checked the double digestion (2 bands seen in the gel) and purified the heavier band from the gel (the linearized plasmid). To confirm the complete double digestion, I then used 1 ul of the purified linearized plasmid in a PCR with M13 primers (0.2 uM end-concentration). No amplification was expected since EagI-HF and NcoI-HF should have removed the sequence between the M13 biding sites of my plasmid. However, the PCR produced a strong band of the exact expected size for the amplification of the sequence between M13 sites.
Does that mean that the double digestion didn’t work at all? Maybe there is a PCR bias favoring the small amount of intact (non-digested) plasmid? In that case, is PCR with M13 primers a valid way to test for complete double digestion of a plasmid?
Thanks in advance,
Dani.
Hi everyone! I performed my very first Gibson assembly (1 vector and 2 fragments) using the NEB Gibson Assembly Cloning Kit (#E5510S) and the assembly efficiency was quite disappointing as revealed by agarose gel electrophoresis. In general, what I’ve seen was that both positive (incubated) and negative (non-incubated) assemblies look the same, which tells me that the assembly efficiency was very low. So, I was wondering if anyone here has experienced the same issue and might be able to give me some “pro-tips” on how to make it work better.
Attached is an overview figure of my experiment and outcomes for reference. Below are the details of the experiment:
(a) On vector preparation: The vector was linearized using double digestion with NcoI-HF (NEB, #R3193S) and EagI-HF (NEB, #R3505S). Both REs display 100% activity in rCutSmart buffer and are not sensitive to procaryotic methylation (i.e., dam or dcm). Reactions were assembly with 1 ug of DNA and 20 U of each enzyme in a 50 ul final volume. Digestions were carried out at 37˚C overnight, following gel verification of successful digestion (final length = 3,920 bp). The digestion was gel purified using the QIAquick PCR & Gel Cleanup Kit (Qiagen, #28506) and eluted in nuclease-free water.
(b) On inserts preparation: insert 1 (494 bp) and insert 2 (2,090 bp) were PCR amplified using primers generating ~40 bp overlapping between fragments (Figure 1a). Amplifications were performed with Q5 High-Fidelity DNA Polymerase (NEB, # M0491S), and gel purified using the QIAquick PCR & Gel Cleanup Kit (Qiagen, #28506) and eluted in nuclease-free water.
(c) On molarity calculations: All concentrations (Figure 1b) were estimated using 2 ul of purified fragments in a Qubit v.2 (Invitrogen). NEB recommends 50-to-100 ng of a vector with a 2-to-3-fold molar excess of each insert (I also have seen some people recommending a 1:1 ratio, do you think it would be better?). I decided to use 60 ng of the vector, and used the following formula to calculate the vector amount (pmols):
pmols = (weight in ng) * 1,000 / (length in bp * 650 Daltons)
Therefore:
pmols = 60 ng * 1,000 / (3,920 bp * 650 Da)
pmols = 0.023
Next, I calculated the necessary mass in ng of each insert for a 1:3 (vector : insert) molar ratio (0.023 pmols of the vector, and 0.07 pmols of the inserts) using the following formula:
(Insert length in Kb / vector length in Kb) * (insert ratio / vector ratio) = insert weight in ng / vector weight in ng
Therefore, for insert 1:
(0.494 Kb / 3.920 Kb) * (3 / 1) = X / 60 ng
0.126 * 3 = X / 60
0.378 * 60 = X
X = 22.6 ng (which gives me 0.07 pmols)
And insert 2:
(2.089 / 3.920) * (3 / 1) = X / 60
X = 96 ng (which gives me 0.07 pmols)
(d) On Gibson assembly: No secrets here, just followed NEB recommendations: Mixed 10 ul of my fragments (see Figure 1b for individual quantities) with 10 ul of 2x Gibson Assembly Master Mix, for a final volume of 20 ul. Reactions were mixed by pipetting and incubated in a thermocycler at 50˚C for 60 min (NEB says that this can be increased to up to 4 hr, have anyone tested this yet?). For control, I used 10 ul of NEBuilder positive control (supplied with the Gibson kit) in a 20 ul reaction. Finally, I included 2 negative controls consisting of my assembly and the NEB control assembly without incubation (left at room temperature ~25˚C during the experiment).
(e) On outcomes (why efficiency so low?): After incubation, I ran 8 ul (equivalent to ~24 ng of the vector) of each positive (mine and NEB positive incubated reactions) and negative (non-incubation reactions) assemblies in a 1% agarose gel (Figure 1c). The result was very disappointing: both positive (incubated) and negative (non-incubated) assemblies look the same, which tells me that the assembly efficiency was very low. Breaking down the bands I can see that:
Experiment: band #1 is the expected size of [vector + insert 1 + insert 2] (6.5Kb), and band #3 of [insert 1 + insert 2] (2.6Kb). I also can see lots of vectors and insert leftovers (bands #2, #4, #5), meaning that the assembly was not efficient, or the molarity ratio should be improved. But most importantly, the incubation (50˚C for 60 min) seems to have little or no impact on the assembly efficiency: What is wrong with that? Because of these results, I decided to not keep going further.
Positive control: As for the NEBbuilder positive control, the bands are very weak in the gel, but I can see a shift in size (from ~3kb to ~3.5Kb) between positive and negative controls (see shift marked as white arrows in Figure 1c). So, I decided to keep going and used 2 ul of the assembly to chemically transform NEB 5-alpha Competent E. coli(High Efficiency) cells (provided with the kit), following NEB protocol. I plated 100 ul of the overgrowth (37˚C for 1 hr) and incubated the plate at 37˚C overnight. The next morning, I could see some colonies, but not many (~27). Do you think the transformation can be improved by using more volume of the assembly (~10 ul) and overgrowth the bacteria for 2 hr instead?
I really appreciate any comments on these results, which might help others in the future. Thanks in advance for your patience and help.
Dani.
