A Novel Solid Artificial Diet for Zeugodacus cucurbitae (Diptera: Tephritidae) Larvae With Fitness Parameters Assessed by Two-Sex Life Table.

The melon fly, Zeugodacus cucurbitae (Coquillett), is a serious pest of many fruits and vegetables throughout the world. Here we have developed an easy and quick-to-prepare solid medium with multiple benefits including reductions in post-rearing waste, storage space, and labor for rearing Z. cucurbitae larvae. The development time from egg to pupa was 19.11 d when larvae were reared on the artificial diet, slightly longer than 17.73 d on pumpkin and 17.13 d on cucumber. Zeugodacus cucurbitae achieved higher values of pupal weight, length, and width on the artificial diet than two natural diet controls. The rates of pupation and adult emergence of Z. cucurbitae grown on the solid medium were comparable with those on pumpkin and cucumber. Furthermore, determined by age-specific two-sex life table method, the age-specific survival rate of Z. cucurbitae was higher on the artificial diet than cucumber but lower than pumpkin. The reproductive ability and population dynamics of Z. cucurbitae were not significantly affected on the solid medium compared with those on the two natural diets. The results suggest that our solid artificial diet is excellent for rearing Z. cucurbitae larvae in laboratory and may be used for its mass rearing, therefore facilitating its research and control.

Isolation and Molecular Characterization of the Transformer Gene From Bactrocera cucurbitae (Diptera: Tephritidae).

transformer (tra) is a switch gene of sex determination in many insects, particularly in Dipterans. However, the sex determination pathway in Bactrocera cucurbitae (Coquillett), a very destructive pest on earth, remains largely uncharacterized. In this study, we have isolated and characterized one female-specific and two male-specific transcripts of the tra gene (Bcutra) of B. cucurbitae. The genomic structure of Bcutra has been determined and the presence of multiple conserved Transformer (TRA)/TRA-2 binding sites in Bcutra has been found. BcuTRA is highly conservative with its homologues in other tephritid fruit flies. Gene expression analysis of Bcutra at different developmental stages demonstrates that the female transcript of Bcutra appears earlier than the male counterparts, indicating that the maternal TRA is inherited in eggs and might play a role in the regulation of TRA expression. The conservation of protein sequence and sex-specific splicing of Bcutra and its expression patterns during development suggest that Bcutra is probably the master gene of sex determination of B. cucurbitae. Isolation of Bcutra will facilitate the development of a genetic sexing strain for its biological control.

The cohesion protein SOLO associates with SMC1 and is required for synapsis, recombination, homolog bias and cohesion and pairing of centromeres in Drosophila Meiosis.

Cohesion between sister chromatids is mediated by cohesin and is essential for proper meiotic segregation of both sister chromatids and homologs. solo encodes a Drosophila meiosis-specific cohesion protein with no apparent sequence homology to cohesins that is required in male meiosis for centromere cohesion, proper orientation of sister centromeres and centromere enrichment of the cohesin subunit SMC1. In this study, we show that solo is involved in multiple aspects of meiosis in female Drosophila. Null mutations in solo caused the following phenotypes: 1) high frequencies of homolog and sister chromatid nondisjunction (NDJ) and sharply reduced frequencies of homolog exchange; 2) reduced transmission of a ring-X chromosome, an indicator of elevated frequencies of sister chromatid exchange (SCE); 3) premature loss of centromere pairing and cohesion during prophase I, as indicated by elevated foci counts of the centromere protein CID; 4) instability of the lateral elements (LE)s and central regions of synaptonemal complexes (SCs), as indicated by fragmented and spotty staining of the chromosome core/LE component SMC1 and the transverse filament protein C(3)G, respectively, at all stages of pachytene. SOLO and SMC1 are both enriched on centromeres throughout prophase I, co-align along the lateral elements of SCs and reciprocally co-immunoprecipitate from ovarian protein extracts. Our studies demonstrate that SOLO is closely associated with meiotic cohesin and required both for enrichment of cohesin on centromeres and stable assembly of cohesin into chromosome cores. These events underlie and are required for stable cohesion of centromeres, synapsis of homologous chromosomes, and a recombination mechanism that suppresses SCE to preferentially generate homolog crossovers (homolog bias). We propose that SOLO is a subunit of a specialized meiotic cohesin complex that mediates both centromeric and axial arm cohesion and promotes homolog bias as a component of chromosome cores.

Homolog pairing and sister chromatid cohesion in heterochromatin in Drosophila male meiosis I.

Drosophila males undergo meiosis without recombination or chiasmata but homologous chromosomes pair and disjoin regularly. The X-Y pair utilizes a specific repeated sequence within the heterochromatic ribosomal DNA blocks as a pairing site. No pairing sites have yet been identified for the autosomes. To search for such sites, we utilized probes targeting specific heterochromatic regions to assay heterochromatin pairing sequences and behavior in meiosis by fluorescence in situ hybridization (FISH). We found that the small fourth chromosome pairs at heterochromatic region 61 and associates with the X chromosome throughout prophase I. Homolog pairing of the fourth chromosome is disrupted when the homolog conjunction complex is perturbed by mutations in SNM or MNM. On the other hand, six tested heterochromatic regions of the major autosomes proved to be largely unpaired after early prophase I, suggesting that stable homolog pairing sites do not exist in heterochromatin of the major autosomes. Furthermore, FISH analysis revealed two distinct patterns of sister chromatid cohesion in heterochromatin: regions with stable cohesion and regions lacking cohesion. This suggests that meiotic sister chromatid cohesion is incomplete within heterochromatin and may occur at specific preferential sites.

CRISPR/Cas9-Mediated Genome Editing System in Insect Genomics and Pest Management.

The CRISPR/Cas9 is being developed as an invaluable system that allows rapid and site-specific genome editing in a wide variety of organisms, including diverse insects. It has been successfully used for gene function annotations of RNAi pathway in insect genomics and will facilitate research on RNAi mechanism. Here, we describe a streamlined method to generate and detect somatic and germline knockout mutations of desired target genes in tephritid pests by injecting mRNA encoding the Cas9 endonuclease and in vitro transcribed single guide RNA (sgRNA) into embryos. Target site selection, sgRNA synthesis, Cas9 synthesis, microinjection, and mutation identification are presented in detail.

Argonaute1 and Gawky Are Required for the Development and Reproduction of Melon fly, Zeugodacus cucurbitae.

Argonaute family genes encode a highly conserved group of proteins that have been associated with RNA silencing in both animals and plants. This study investigates the importance of microRNA biogenesis key regulators Argonaute1 (Ago1) and Gawky genes in the post-embryonic and ovarian development of the melon fly, Zeugodacus cucurbitae. The expression levels of these genes were mapped in all developmental stages and different adult tissues. Their roles in development were investigated using RNA interference (RNAi) via two different dsRNA delivery techniques. Embryo microinjection and oral feeding of third instar larvae successfully knocked down and greatly reduced the expression level of the target genes. Additionally, ex vivo essays revealed the stability of dsRNA in food was sufficient for gene silencing, although its integrity was affected in midgut. A wide range of phenotypes were observed on pupation, segmentation, pigmentation, and ovarian development. RNAi-mediated silencing of Gawky caused high mortality and loss of body segmentation, while Ago1 knockdown affected ovarian development and pigmentation. Developmental abnormalities and ovarian malformations caused by silencing these genes suggest that these genes are crucial for viability and reproductive capacity of Z. cucurbitae, and may be used as potential target genes in pest management.

Efficient somatic and germline genome engineering of Bactrocera dorsalis by the CRISPR/Cas9 system.

BACKGROUND: Bactrocera dorsalis (Hendel), a very destructive insect pest of many fruits and vegetables, is widespread in many Asian countries. To facilitate control of this pest, it is essential to investigate its genetics and gene function using targeted gene disruption. RESULTS: Here, we describe successful targeted mutagenesis of the white and transformer genes in B. dorsalis through use of the clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) system. Co-injection of the white sgRNA and Cas9 mRNA into B. dorsalis embryos caused eye color change, and the white mutations in the germline were heritable. CRISPR-mediated knockout of the sex determination gene transformer (tra) in B. dorsalis resulted in a male-biased sex ratio and adult flies with abnormal outer and interior reproductive organs. Small indels and substitutions were induced by CRIRPR for both genes. CONCLUSION: Our data demonstrate that somatic and germline genome engineering of the pest B. dorsalis can be performed efficiently using the CRISPR/Cas9 system, opening the door to the use of the CRISPR-mediated method for functional annotations of genes in B. dorsalis and for its population control using, for example, such as gene drive. © 2018 Society of Chemical Industry.

Targeting Inhibition of SmpB by Peptide Aptamer Attenuates the Virulence to Protect Zebrafish against Aeromonas veronii Infection.

Aeromonas veronii is an important pathogen of aquatic animals, wherein Small protein B (SmpB) is required for pathogenesis by functioning as both a component in stalled-ribosome rescue and a transcription factor in upregulation of virulence gene bvgS expression. Here a specific peptide aptamer PA-1 was selected from peptide aptamer library by bacterial two-hybrid system employing pBT-SmpB as bait. The binding affinity between SmpB and PA-1 was evaluated using enzyme-linked immunosorbent assay. The key amino acids of SmpB that interact with PA-1 were identified. After PA-1 was introduced into A. veronii, the engineered strain designated as A. veronii (pN-PA-1) was more sensitive and grew slower under salt stress in comparison with wild type, as the disruption of SmpB by PA-1 resulted in significant transcription reductions of virulence-related genes. Consistent with these observations, A. veronii (pN-PA-1) was severely attenuated in model organism zebrafish, and vaccination of zebrafish with A. veronii (pN-PA-1) induced a strong antibody response. The vaccinated zebrafish were well protected against subsequent lethal challenges with virulent parental strain. Collectively, we propose that targeting inhibition of SmpB by peptide aptamer PA-1 possesses the desired qualities for a live attenuated vaccine against pathogenic A. veronii.

Sisters unbound is required for meiotic centromeric cohesion in Drosophila melanogaster.

Regular meiotic chromosome segregation requires sister centromeres to mono-orient (orient to the same pole) during the first meiotic division (meiosis I) when homologous chromosomes segregate, and to bi-orient (orient to opposite poles) during the second meiotic division (meiosis II) when sister chromatids segregate. Both orientation patterns require cohesion between sister centromeres, which is established during meiotic DNA replication and persists until anaphase of meiosis II. Meiotic cohesion is mediated by a conserved four-protein complex called cohesin that includes two structural maintenance of chromosomes (SMC) subunits (SMC1 and SMC3) and two non-SMC subunits. In Drosophila melanogaster, however, the meiotic cohesion apparatus has not been fully characterized and the non-SMC subunits have not been identified. We have identified a novel Drosophila gene called sisters unbound (sunn), which is required for stable sister chromatid cohesion throughout meiosis. sunn mutations disrupt centromere cohesion during prophase I and cause high frequencies of non-disjunction (NDJ) at both meiotic divisions in both sexes. SUNN co-localizes at centromeres with the cohesion proteins SMC1 and SOLO in both sexes and is necessary for the recruitment of both proteins to centromeres. Although SUNN lacks sequence homology to cohesins, bioinformatic analysis indicates that SUNN may be a structural homolog of the non-SMC cohesin subunit stromalin (SA), suggesting that SUNN may serve as a meiosis-specific cohesin subunit. In conclusion, our data show that SUNN is an essential meiosis-specific Drosophila cohesion protein.

Meiosis in male Drosophila.

Meiosis entails sorting and separating both homologous and sister chromatids. The mechanisms for connecting sister chromatids and homologs during meiosis are highly conserved and include specialized forms of the cohesin complex and a tightly regulated homolog synapsis/recombination pathway designed to yield regular crossovers between homologous chromatids. Drosophila male meiosis is of special interest because it dispenses with large segments of the standard meiotic script, particularly recombination, synapsis and the associated structures. Instead, Drosophila relies on a unique protein complex composed of at least two novel proteins, SNM and MNM, to provide stable connections between homologs during meiosis I. Sister chromatid cohesion in Drosophila is mediated by cohesins, ring-shaped complexes that entrap sister chromatids. However, unlike other eukaryotes Drosophila does not rely on the highly conserved Rec8 cohesin in meiosis, but instead utilizes two novel cohesion proteins, ORD and SOLO, which interact with the SMC1/3 cohesin components in providing meiotic cohesion.

SOLO: a meiotic protein required for centromere cohesion, coorientation, and SMC1 localization in Drosophila melanogaster.

Sister chromatid cohesion is essential to maintain stable connections between homologues and sister chromatids during meiosis and to establish correct centromere orientation patterns on the meiosis I and II spindles. However, the meiotic cohesion apparatus in Drosophila melanogaster remains largely uncharacterized. We describe a novel protein, sisters on the loose (SOLO), which is essential for meiotic cohesion in Drosophila. In solo mutants, sister centromeres separate before prometaphase I, disrupting meiosis I centromere orientation and causing nondisjunction of both homologous and sister chromatids. Centromeric foci of the cohesin protein SMC1 are absent in solo mutants at all meiotic stages. SOLO and SMC1 colocalize to meiotic centromeres from early prophase I until anaphase II in wild-type males, but both proteins disappear prematurely at anaphase I in mutants for mei-S332, which encodes the Drosophila homologue of the cohesin protector protein shugoshin. The solo mutant phenotypes and the localization patterns of SOLO and SMC1 indicate that they function together to maintain sister chromatid cohesion in Drosophila meiosis.