Polyandrous Mexican Fruit Flies: Second Male Paternity and Biological Attributes of Transgenic Strains

Anastrepha ludens (Diptera: Tephritidae), is a damaging agricultural pest. Currently, the Sterile Insect Technique (SIT) is used as part of its control. The SIT consists of the mass-rearing, sterilization, and release of insects in target areas. Sterile males mate with wild females, and prevent them from laying fertile eggs. However, even if females mate with sterile males, they can then remate with a second male. If this second male is wild, then this could reduce the efficiency of the SIT by producing viable offspring. The amount of progeny produced by second males (P2 values) for A. ludens is unknown. Here, we evaluated the biological attributes, mating competitiveness, and the proportion of male paternity gained by the second male, using strains that carry fluorescent marker genes and can be potentially used to develop transgenic sexing strains. Furthermore, the transgenic strains were irradiated, to test their ability to induce sterility in females. We found that the 443-G strain had significantly higher larval survival than the 419-R strain. No significant difference was found between the two strains in their mating probability with wild females. We found P2 values between 67 and 74% for the 419-R and the 443-G strain, respectively. Second male sperm precedence only decreased slightly after 12 days, suggesting that sperm from the first and second male is not mixing with time, but rather the second male’s sperm prevails. Furthermore, sterile 443-G males induced significantly higher sterility in females than sterile males from the 419-R strain. The apparent lower ability of the 443-G strain to inhibit female remating should be further investigated. Knowledge of the pre and postcopulatory performance of transgenic strains will help in understanding their potential for control.

A Drosophila Toolkit for Imaging of HA-tagged Proteins Unveiled a Block in Autophagy Flux in the Last Instar Larval Fat Body

For in vivo functional analysis of a protein of interest (POI), multiple transgenic strains with POI harboring different tags are needed but generation of these strains is still labor-intensive work. To overcome this, we developed a versatile Drosophila toolkit with a genetically encoded single-chain variable fragment for the HA epitope tag: HA Frankenbody. This system allows various analyses of HA-tagged POI in live tissues by simply crossing an HA Frankenbody fly with an HA-tagged POI fly. Strikingly, the GFP-mCherry tandem fluorescent-tagged HA Frankenbody revealed a block in autophagic flux and an accumulation of enlarged autolysosomes in the last instar larval and prepupal fat body. Autophagy was dispensable for the swelling of lysosomes, indicating that lysosomal activity is downregulated at this stage. Furthermore, forced activation of lysosomes by fat body-targeted overexpression of Mitf, the single MiTF/TFE family gene in Drosophila, suppressed the lysosomal swelling and resulted in pupal lethality. Collectively, we propose that downregulated lysosomal function in the fat body plays a role in the metamorphosis of Drosophila.

Auxin treatment increases lifespan in Caenorhabditis elegans

ABSTRACT The auxin-inducible degradation system (AID) has proven to be a highly versatile technology for rapid, robust and reversible depletion of proteins in multiple model systems. In recent years, AID has been adapted into the nematode Caenorhabditis elegans as a tool for conditional protein knockdown. Numerous transgenic strains have been created that, upon auxin exposure, undergo protein inactivation in the worm germline or somatic tissues, both during development and in young adults. Since longevity assays often involve long-term gene- and protein-manipulation, the facility for spatiotemporally precise and extended protein removal makes AID a potentially highly valuable tool for aging biology. However, whether auxins themselves impact worm longevity has not been directly addressed. Here, we show that prolonged exposure to indole 3-acetic acid (IAA), the auxin used in worm AID studies, extends lifespan. We also report that two transgenic strains expressing Arabidopsis proteins that are key components of the AID platform are longer lived than wild-type animals. Together, our results highlight the necessity for exercising caution while utilizing AID for longevity studies and in interpreting the resulting data. This article has an associated First Person interview with the first author of the paper.

Overexpression of the Transcription Factor AtLEC1 Significantly Improved the Lipid Content of Chlorella ellipsoidea

Microalgae are considered to be a highly promising source for the production of biodiesel. However, the regulatory mechanism governing lipid biosynthesis has not been fully elucidated to date, and the improvement of lipid accumulation in microalgae is essential for the effective production of biodiesel. In this study,LEAFY COTYLEDON1 (LEC1)fromArabidopsis thaliana, a transcription factor (TF) that affects lipid content, was transferred intoChlorella ellipsoidea. Compared with wild-type (WT) strains, the total fatty acid content and total lipid content ofAtLEC1transgenic strains were significantly increased by 24.20–32.65 and 22.14–29.91%, respectively, under mixotrophic culture conditions and increased by 24.4–28.87 and 21.69–30.45%, respectively, under autotrophic conditions, while the protein content of the transgenic strains was significantly decreased by 18.23–21.44 and 12.28–18.66%, respectively, under mixotrophic and autotrophic conditions. Fortunately, the lipid and protein content variation did not affect the growth rate and biomass of transgenic strains under the two culture conditions. According to the transcriptomic data, the expression of 924 genes was significantly changed in the transgenic strain (LEC1-1). Of the 924 genes, 360 were upregulated, and 564 were downregulated. Based on qRT-PCR results, the expression profiles of key genes in the lipid synthesis pathway, such asACCase,GPDH,PDAT1, andDGAT1, were significantly changed. By comparing the differentially expressed genes (DEGs) regulated byAtLEC1inC. ellipsoideaandArabidopsis, we observed that approximately 59% (95/160) of the genes related to lipid metabolism were upregulated inAtLEC1transgenicChlorella. Our research provides a means of increasing lipid content by introducing exogenous TF and presents a possible mechanism ofAtLEC1regulation of lipid accumulation inC. ellipsoidea.

Using Bacillus subtilis as a Host Cell to Express an Antimicrobial Peptide from the Marine Chordate Ciona intestinalis

Ciona molecule against microbes-A24 (CiMAM) isolated from the marine chordate Ciona intestinalis is an antimicrobial peptide. To generate CiMAM-expressing transgenic Bacillus subtilis, we constructed a plasmid expressing recombinant CiMAM (rCiMAM) and introduced it into B. subtilis. Transgenic strains C117 and C166 were selected since they were able to highly and stably express rCiMAM. We studied the bactericidal activity of pepsin-digested extracts from rCiMAM-expressing strains against freshwater and euryhaline pathogens that commonly occur in aquaculture ponds and found no difference from that of lactoferricin-expressing strains. The bactericidal activity of 1-μL aliquot from a total 5.5 mL extracted from 5 mL of cultured C117 (1.45 × 108 CFU·mL−1) and C166 (2.17 × 108 CFU·mL−1) against halophilic bacteria was equivalent to the efficacy of 57.06 and 32.35 ng of Tetracycline against Vibrio natriegens, 47.07 and 25.2 ng against V. parahaemolyticus, and 58.17 and 36.55 ng against V. alginolyticus, respectively, indicating higher bactericidal activity of pepsin-extracts from rCiMAM-containing strains against halophilic bacteria compared to that from lactoferricin-containing strains. Since the antibacterial activity of rCiMAM-expressing B. subtilis strains shows higher competence against halophilic pathogens compared to that against freshwater and euryhaline pathogens, these strains are promising candidates to protect marine fish and shellfish from halophilic bacterial infection.

Transformation and functional verification of Cry5Aa in cotton

Most of the cotton bollworm-resistant genes applied in cotton are more than 20 years and they all belong to Cry1Ab/c family, but the insect-resistant effects of Cry5Aa on cotton were rarely reported. The possible risk of resistance is increasing. The study synthesized a novel bollworm-resistant gene Cry5Aa artificially based on preferences of cotton codon. The new gene was transferred to cotton through the method of pollen tube pathway. The transgenic strains were identified by kanamycin test in field and laboratory PCR analysis. Meanwhile, an insect resistance test was conducted by artificial bollworm feeding with transgenic leaves and GK19 was used as a control in this study. Results showed that rate of positive transgenic strains with kanamycin resistance in the first generation (T1), the second generation (T2) and the third generation (T3) respectively were 7.76%, 73.1% and 95.5%. However, PCR analysis showed that the positive strain rate in T1, T2 and T3 were 2.35%, 55.8% and 94.5%, respectively. The resistant assay of cotton bollworm showed that the mortality rate of the second, third and fourth instar larva feed by the transgenic cotton leaves, were 85.42%, 73.35% and 62.79%, respectively. There was a significant difference between transgenic plant of Cry5Aa and GK19 in insect resistance. Finally, we also conducted the further analysis of gene expression patterns, gene flow and the effect on non-target pest in the study. The results showed that Cry5Aa gene had less environmental impact, and Cry5Aa has been transferred successfully and expressed stably in cotton. Therefore, the novel bollworm resistance gene can partially replace the current insect-resistance gene of Lepidoptera insects.

Optimization of CRISPR/Cas System for Improving Genome Editing Efficiency in Plasmodium falciparum

Studies of molecular mechanisms and related gene functions have long been restricted by limited genome editing technologies in malaria parasites. Recently, a simple and effective genome editing technology, the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system, has greatly facilitated these studies in many organisms, including malaria parasites. However, due to the special genome feature of malaria parasites, the manipulation and gene editing efficacy of the CRISPR/Cas system in this pathogen need to be improved, particularly in the human malaria parasite, Plasmodium falciparum. Herein, based on the CRISPR/Cas9 system, we developed an integrating strategy to generate a Cas9i system, which significantly shortened the time for generation of transgenic strains in P. falciparum. Moreover, with this Cas9i system, we have successfully achieved multiplexed genome editing (mutating or tagging) by a single-round transfection in P. falciparum. In addition, we for the first time adapted AsCpf1 (Acidaminococcus sp. Cpf1), an alternative to Cas9, into P. falciparum parasites and examined it for gene editing. These optimizations of the CRISPR/Cas system will further facilitate the mechanistic research of malaria parasites and contribute to eliminating malaria in the future.

Caenorhabditis elegans Models to Investigate the Mechanisms Underlying Tau Toxicity in Tauopathies

The understanding of the genetic, biochemical, and structural determinants underlying tau aggregation is pivotal in the elucidation of the pathogenic process driving tauopathies and the design of effective therapies. Relevant information on the molecular basis of human neurodegeneration in vivo can be obtained using the nematode Caenorhabditis elegans (C. elegans). To this end, two main approaches can be applied: the overexpression of genes/proteins leading to neuronal dysfunction and death, and studies in which proteins prone to misfolding are exogenously administered to induce a neurotoxic phenotype. Thanks to the easy generation of transgenic strains expressing human disease genes, C. elegans allows the identification of genes and/or proteins specifically associated with pathology and the specific disruptions of cellular processes involved in disease. Several transgenic strains expressing human wild-type or mutated tau have been developed and offer significant information concerning whether transgene expression regulates protein production and aggregation in soluble or insoluble form, onset of the disease, and the degenerative process. C. elegans is able to specifically react to the toxic assemblies of tau, thus developing a neurodegenerative phenotype that, even when exogenously administered, opens up the use of this assay to investigate in vivo the relationship between the tau sequence, its folding, and its proteotoxicity. These approaches can be employed to screen drugs and small molecules that can interact with the biogenesis and dynamics of formation of tau aggregates and to analyze their interactions with other cellular proteins.

Involvement of green technology in microalgal biodiesel production

According to the report of the renewable energy policy network for the 21st century published in 2014, biodiesel and bioethanol are the most used biofuels and are responsible for transportation worldwide. Biodiesel specially has shown an increase in production globally by 15 times by volume from 2002 to 2012. Promising feedstock of biodiesel are cyanobacteria and microalgae as they possess a shorter cultivation time (4 fold lesser) and high oil content (10 fold higher) than corn, jatropha and soybean (conventional oil-producing territorial plants). Various valuable natural chemicals are also produced from these organisms including food supplements such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), pigments, and vitamins. Additionally, cellular components of microalgae and cyanobacteria are connected with therapeutic characteristics such as anti-inflammatory, antioxidant, antiviral and immune stimulating. Commercialization of algal biodiesel (or other products) can be achieved by isolating and identifying the high-yielding strains that possess a faster growth rate. Indigenous strains can be genetically engineered into high-yielding transgenic strains. The present article discusses about the use of nanotechnology and genetic engineering approach for improved lipid accumulation in microalgae for biodiesel production.

Heterologous phytase expression in the food filamentous fungus Aspergillus oryzae using the added rice husk cultivation model

Aspergillus oryzae, a safe filamentous fungus, is widely used in food and enzyme production. In this study, we examined a cultivation model using rice husks as carrier to assess the capacity of recombinant protein production in A. oryzae. The model was first tested with the A. oryzae strain expressing the DsRed reporter gene. Expression of DsRed was easily detected by the pink color of the fungal mycelium on culture media and under a fluorescence microscope. The model was then evaluated with the phyA gene encoding a phytase from the fungus Aspergillus fumigatus. The phyA expression cassette regulated by the amyB promoter was permanently integrated into the genome of A. oryzae via Agrobacterium tumefaciens-mediated transformation with the pyrG nutritional marker. Results showed that A. oryzae transgenic strains carrying 2−3 copies of the phyA gene in their genomes exhibited a significant increase in phytase activity on agar medium supplemented with phytate. With rice husks added, these transgenic strains could secrete the recombinant phytase into the culture and phytase activity of the crude enzyme solution increased by 4.3 times compared to the unstransgenic A. oryzae. The established cultivation model and the transgenic approach in this study represent a potential for being used in production of secreted recombinant enzymes for animal feeds.