Developing CRISPR-based sex-ratio distorters for the genetic control of fruit fly pests: A how to manual

Agricultural pest control using genetic-based methods provides a species-specific and environmentally harmless way for population suppression of fruit flies. One way to improve the efficiency of such methods is through self-limiting, female-eliminating approaches that can alter an insect populations' sex ratio toward males. In this microreview, we summarize recent advances in synthetic sex ratio distorters based on X-chromosome shredding that can induce male-biased progeny. We outline the basic principles to guide the efficient design of an X-shredding system in an XY heterogametic fruit fly species of interest using CRISPR/Cas gene editing, newly developed computational tools, and insect genetic engineering. We also discuss technical aspects and challenges associated with the efficient transferability of this technology in fruit fly pest populations, toward the potential use of this new class of genetic control approaches for pest management purposes.     

EBNA3C Directs Recruitment of RBPJ (CBF1) to Chromatin during the Process of Gene Repression in EBV Infected B Cells

It is well established that Epstein-Barr virus nuclear antigen 3C (EBNA3C) can act as a potent repressor of gene expression, but little is known about the sequence of events occurring during the repression process. To explore further the role of EBNA3C in gene repression–particularly in relation to histone modifications and cell factors involved–the three host genes previously reported as most robustly repressed by EBNA3C were investigated. COBLL1, a gene of unknown function, is regulated by EBNA3C alone and the two co-regulated disintegrin/metalloproteases, ADAM28 and ADAMDEC1 have been described previously as targets of both EBNA3A and EBNA3C. For the first time, EBNA3C was here shown to be the main regulator of all three genes early after infection of primary B cells. Using various EBV-recombinants, repression over orders of magnitude was seen only when EBNA3C was expressed. Unexpectedly, full repression was not achieved until 30 days after infection. This was accurately reproduced in established LCLs carrying EBV-recombinants conditional for EBNA3C function, demonstrating the utility of the conditional system to replicate events early after infection. Using this system, detailed chromatin immunoprecipitation analysis revealed that the initial repression was associated with loss of activation-associated histone modifications (H3K9ac, H3K27ac and H3K4me3) and was independent of recruitment of polycomb proteins and deposition of the repressive H3K27me3 modification, which were only observed later in repression. Most remarkable, and in contrast to current models of RBPJ in repression, was the observation that this DNA-binding factor accumulated at the EBNA3C-binding sites only when EBNA3C was functional. Transient reporter assays indicated that repression of these genes was dependent on the interaction between EBNA3C and RBPJ. This was confirmed with a novel EBV-recombinant encoding a mutant of EBNA3C unable to bind RBPJ, by showing this virus was incapable of repressing COBLL1 or ADAM28/ADAMDEC1 in newly infected primary B cells.     

Does fragmented river connectivity alter the reproductive behavior of the potamodromous fish Alburnus vistonicus?

Blocked riverine spawning migration routes constitute one of the main threats of potamodromous fish that require well-connected habitats of high quality to maintain their unique life-history form. Alburnus vistonicus, a potamodromous species inhabiting the Vistonis freshwater system in northern Greece, manages to cope with wide salinity fluctuations in Vistonis Lake, but the impact of the fragmented connectivity of the lake’s two main freshwater suppliers, Kosynthos and Kompsatos rivers, has not been assessed. We analyzed the species whole reproductive cycle and we divided it into phases. Cyclicity was observed in gonadosomatic index and oocyte diameter, while total fecundity remained constant prior to the onset of the spawning season. Stomach fullness and condition factor differed among the surveyed habitats. Successful spawning and multiple spawning were verified, as well as spawning migration and potamodromy. However, mature females with markers of recent spawning activity were captured also in the lake, suggesting spawning occurrence in the lake or a previously undocumented oscillatory behavior between the lake and the rivers during the spawning season and between sequential spawning events. Either behavior may serve as a mechanism to overcome the restricted spawning migration routes due to the fragmented river connectivity.

     

Characterization of purified leaf cytosolic pyruvate kinase from the C4 plant Cynodon dactylon

Cytosolic pyruvate kinase (EC 2.7.1.40) from leaves of the C₄ plant Cynodon dactylon (L.) Pers. was purified 56-fold to apparent homogeneity by polyethylene glycol fractionation and column chromatography including Q-Sepharose anion exchanger, ADP-Agarose and gel filtration. Nondenaturing PAGE of the final preparation resulted in a single protein band that co-migrated with the pyruvate kinase activity. Gel filtration and SDS-PAGE (± DTT) showed that this enzyme has a molecular mass of 200 kDa and is a homotetramer with a subunit molecular mass of 50 kDa. The subunits are not associated to each other with S-S bonds. The enzyme has a pH optimum of 6.2 and is heat stable. Typical Michaelis-Menten kinetics was obtained for both substrates, PEP and ADP, with K_m values of 64 and 235 μ M , respectively. Initial velocity studies indicated a sequential binding of the substrates to the enzyme.     

Evolutionary species–area curves as revealed by single-island endemics: insights for the inter-provincial species–area relationship

The species–area relationship (SAR) between different biological provinces is one of the most interesting, but least explored aspects of the well-known species–area pattern. Following the usage that a biological province is a region whose species have for the most part evolved within it, rather than immigrating from somewhere else, we propose that islands can be considered equivalent to biological provinces for single island endemic species (SIEs). Hence, analyses of the relationships between numbers of SIEs and island area can be used as model systems to explore the form of inter-provincial SARs. We analyzed 13 different data sets derived from 11 sources, using the power (log–log) model. Eleven of the SIE–area relationships were statistically significant, explaining high proportions of the variance in SIE numbers (R² 0.57–0.95). The z-values (slopes) of the statistically significant SIE–area relationships ranged from 0.47 to 1.13, with a mean value of 0.80 (SD±0.24).
All the island systems in which SIE represent >50% of species exhibited z-values for the SARs of native species higher than those deemed typical of archipelagic SARs. The SIE–area slopes are quite similar to those previously calculated for inter-provincial SARs, indicating that, when speciation becomes the dominant process adding to the species richness of assemblages, high z-values should be anticipated, regardless of the biogeographical scale of the study system.     

Nuclear and Wolbachia-based multimarker approach for the rapid and accurate identification of tsetse species

Background

Tsetse flies (Diptera: Glossinidae) are solely responsible for the transmission of African trypanosomes, causative agents of sleeping sickness in humans and nagana in livestock. Due to the lack of efficient vaccines and the emergence of drug resistance, vector control approaches such as the sterile insect technique (SIT), remain the most effective way to control disease. SIT is a species-specific approach and therefore requires accurate identification of natural pest populations at the species level. However, the presence of morphologically similar species (species complexes and sub-species) in tsetse flies challenges the successful implementation of SIT-based population control.

Results

In this study, we evaluate different molecular tools that can be applied for the delimitation of different Glossina species using tsetse samples derived from laboratory colonies, natural populations and museum specimens. The use of mitochondrial markers, nuclear markers (including internal transcribed spacer 1 (ITS1) and different microsatellites), and bacterial symbiotic markers (Wolbachia infection status) in combination with relatively inexpensive techniques such as PCR, agarose gel electrophoresis, and to some extent sequencing provided a rapid, cost effective, and accurate identification of several tsetse species.

Conclusions

The effectiveness of SIT benefits from the fine resolution of species limits in nature. The present study supports the quick identification of large samples using simple and cost effective universalized protocols, which can be easily applied by countries/laboratories with limited resources and expertise.