Sterile Insect Research Articles

Characterization of the Drosophila suzukii β2-tubulin gene and the utilization of its promoter to monitor sex separation and insemination.

The Drosophila melanogaster β2-tubulin gene (Dm-β2t) controls the function of microtubules in the testis and sperm, and has been evaluated for use in biocontrol strategies based on the sterile insect technique, including sexing and the induction of male sterility. The spotted-wing Drosophila (Drosophila suzukii) is native to eastern Asia but has spread globally as an invasive pest of fruit crops, so biocontrol strategies are urgently required for this species. We therefore isolated the β2tubulin ortholog Ds-β2t from the USA laboratory strain of D. suzukii and confirmed the presence of functional motifs by aligning orthologs from multiple insects. The developmental expression profile of Ds-β2t was determined by RT-PCR using gene-specific primers and was similar to that of Dm-β2t. We then isolated the Ds-β2t promoter and used it to generate transgenic strains expressing a testis-specific fluorescent protein starting from the thirdinstar larvae. Efficient sexing was achieved based on fluorescence detection, and the transgenic males showed a similar survival rate to wild-type males. Fluorescence imaging and PCR were also used to confirm the insemination of wild-type females by transgenic males. We therefore confirm that D. suzukii strains expressing fluorescent markers under the control of the Ds-β2t promoter can be used for sexing and the confirmation of mating, and we discuss the wider potential of the Ds-β2t promoter in the context of genetic control strategies for D. suzukii.

Effects of Gamma Irradiation on Mating Competitiveness of Male <em>Culex quinquefasciatus</em> (Diptera:Culicidae) in Laboratory

Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:"Times New Roman","serif";} Culex quinquefasciatus is the main vector of lymphatic filariasis in Pekalongan City. Sterile Insect Technique (SIT) can be employed as complementary vector control for filariasis. The key success of this technique depends on the ability of laboratory-reared sterile males to mate with the wild-type females. This research aimed to was to determine the mating competitiveness, fecundity and fertility of sterile males of Cx. quinquefasciatus. The pupae of Cx. quinquefasciatus were gamma irradiated at doses of 60, 70, and 80 Gy, whereas unirradiated pupae were prepared as control. The mosquitoes emerging from the irradiated pupae were found to be able to mate with normal females in the cages. Observation was done for the mean female laying eggs, the fecundity, the fertility and the mating competitiveness. The observation data were analyzed by one-way ANOVA. The results show that the irradiated Cx. quinquefasciatus at the test doses does not affect the fecundity and the mating competitiveness, but the fertility is disturbed (sterile) . A dose of 70 Gy was found to be the optimum dose, which gave a fertility rate of 1.8 % (98.2 % sterile) with a value of competitiveness (C index) of 0.568. Based on these results, irradiated Cx. quinquefasciatus can be recommended for semifield application.

Identification and in situ hybridization to mitotic chromosomes of a molecular marker linked to maleness in Anastrepha fraterculus (Wied.)

The present report shows the molecular identification, isolation and citologically localization of a DNA-sequence from the South American fruit fly Anastrepha fraterculus (DIPTERA: Tephritidae) involved in sex- determination. It belongs to the Tephritidae family, the true fruit flies which are consider a pest of fruit crops. The sex determination system is of vital importance in the genetic control of the fruit fly pest: Sterile Insect Technique which unlike chemical control tactics, is environmentally friendly and does not pose any health concerns. We used in situ hybridization on mitotic chromosomes for localizing the primary sex determination factor in this fruit fly pest. Our results show that in Anastrepha fraterculus the Y chromosome is responsible for sex determination

Comparative genomics of Klebsiella michiganensis BD177 and related members of Klebsiella sp. reveal the symbiotic relationship with Bactrocera dorsalis

BackgroundBactrocera dorsalis is a destructive polyphagous and highly invasive insect pest of tropical and subtropical species of fruit and vegetable crops. The sterile insect technique (SIT) has been used for decades to control insect pests of agricultural, veterinary, and human health importance. Irradiation of pupae in SIT can reduce the ecological fitness of the sterile insects. Our previous study has shown that a gut bacterial strain BD177 that could restore ecological fitness by promoting host food intake and metabolic activities.ResultsUsing long-read sequence technologies, we assembled the complete genome of K. michiganensis BD177 strain. The complete genome of K. michiganensis BD177 comprises one circular chromosome and four plasmids with a GC content of 55.03%. The pan-genome analysis was performed on 119 genomes (strain BD177 genome and 118 out of 128 published Klebsiella sp. genomes since ten were discarded). The pan-genome includes a total of 49305 gene clusters, a small number of 858 core genes, and a high number of accessory (10566) genes. Pan-genome and average nucleotide identity (ANI) analysis showed that BD177 is more similar to the type strain K. michiganensis DSM2544, while away from the type strain K. oxytoca ATCC13182. Comparative genome analysis with 21 K. oxytoca and 12 K. michiganensis strains, identified 213 unique genes, several of them related to amino acid metabolism, metabolism of cofactors and vitamins, and xenobiotics biodegradation and metabolism in BD177 genome.ConclusionsPhylogenomics analysis reclassified strain BD177 as a member of the species K. michiganensis. Comparative genome analysis suggested that K. michiganensis BD177 has the strain-specific ability to provide three essential amino acids (phenylalanine, tryptophan and methionine) and two vitamins B (folate and riboflavin) to B. dorsalis. The clear classification status of BD177 strain and identification of unique genetic characteristics may contribute to expanding our understanding of the symbiotic relationship of gut microbiota and B. dorsalis.

Separating two tightly linked species-defining phenotypes in Bactrocera with hybrid recombinant analysis

BackgroundBactrocera tryoni and Bactrocera neohumeralis mate asynchronously; the former mates exclusively around dusk while the latter mates during the day. The two species also differ in the colour of the post-pronotal lobe (callus), which is predominantly yellow in B. tryoni and brown in B. neohumeralis. We have examined the genetic relationship between the two characters in hybrids, backcrosses and multigeneration hybrid progeny.ResultsOur analysis of the mating time of the parental species revealed that while B. tryoni mate exclusively at dusk, B. neohumeralis females pair with B. neohumeralis males during the day and with B. tryoni males at dusk. We found considerable variance in mating time and callus colour among hybrid backcross individuals of both sexes but there was a strong although not invariant trend for callus colour to co-segregate with mating time in both sexes. To genetically separate these two phenotypes we allowed the interspecific F1 hybrids to propagate for 25 generations (F25) without selection for mating time or callus colour, finding that the advanced hybrid population had moved towards B. tryoni phenotypes for both traits. Selection for day mating in replicate lines at F25 resulted in significant phenotypic shifts in both traits towards B. neohumeralis phenotypes in F26. However, we were unable to completely recover the mating time profile of B. neohumeralis and relaxation of selection for day mating led to a shift back towards dusk mating, but not yellow callus colour, by F35.ConclusionWe conclude that the inheritance of the two major species-defining traits is separable but tightly linked and involves more than one gene in each case. It also appears that laboratory conditions select for the B. tryoni phenotypes for mating time. We discuss our findings in relation to speciation theory and the likely effects of domestication during the generation of mass release strains for sterile insect control programmes.

Climate stress resistance in male Queensland fruit fly varies among populations of diverse geographic origins and changes during domestication

BackgroundThe highly polyphagous Queensland fruit fly (Bactrocera tryoni Froggatt) expanded its range substantially during the twentieth century and is now the most economically important insect pest of Australian horticulture, prompting intensive efforts to develop a Sterile Insect Technique (SIT) control program. Using a “common garden” approach, we have screened for natural genetic variation in key environmental fitness traits among populations from across the geographic range of this species and monitored changes in those traits induced during domestication.ResultsSignificant variation was detected between the populations for heat, desiccation and starvation resistance and wing length (as a measure of body size). Desiccation resistance was correlated with both starvation resistance and wing length. Bioassay data for three resampled populations indicate that much of the variation in desiccation resistance reflects persistent, inherited differences among the populations. No latitudinal cline was detected for any of the traits and only weak correlations were found with climatic variables for heat resistance and wing length. All three stress resistance phenotypes and wing length changed significantly in certain populations with ongoing domestication but there was also a strong population by domestication interaction effect for each trait.ConclusionsEcotypic variation in heat, starvation and desiccation resistance was detected in Australian Qfly populations, and these stress resistances diminished rapidly during domestication. Our results indicate a need to select source populations for SIT strains which have relatively high climatic stress resistance and to minimise loss of that resistance during domestication.

Development and characterization of a pupal-colour based genetic sexing strain of Anastrepha fraterculus sp. 1 (Diptera: Tephritidae)

BackgroundArea-wide integrated pest management programs (AW-IPM) incorporating sterile insect technique (SIT) have been successful in suppressing populations of different fruit fly species during the last six decades. In addition, the development of genetic sexing strains (GSS) for different fruit fly species has allowed for sterile male-only releases and has significantly improved the efficacy and cost effectiveness of the SIT applications. The South American Fruit Fly Anastrepha fraterculus (Diptera: Tephritidae) is a major agricultural pest attacking several fruit commodities. This impedes international trade and has a significant negative impact on the local economies. Given the importance of sterile male-only releases, the development of a GSS for A. fraterculus would facilitate the implementation of an efficient and cost-effective SIT operational program against this insect pest species.ResultsFor potential use in a GSS, three new morphological markers (mutants) were isolated in a laboratory strain of A. fraterculus sp. 1, including the black pupae (bp) gene located on chromosome VI. The black pupa phenotype was used as a selectable marker to develop genetic sexing strains by linking the wild type allele (bp+) to the Y-chromosome -via irradiation to induce a reciprocal Y-autosome translocation. Four GSS were established and one of them, namely GSS-89, showed the best genetic stability and the highest fertility. This strain was selected for further characterization and cytogenetic analysis.ConclusionsWe herein report the development of the first genetic sexing strain of a major agricultural pest, A. fraterculus sp. 1, using as a selectable marker the black pupae genetic locus.

Identification and characterization of four Drosophila suzukii cellularization genes and their promoters

BackgroundThe spotted-wing Drosophila (Drosophila suzukii) is a widespread invasive pest that causes severe economic damage to fruit crops. The early development of D. suzukii is similar to that of other Drosophilids, but the roles of individual genes must be confirmed experimentally. Cellularization genes coordinate the onset of cell division as soon as the invagination of membranes starts around the nuclei in the syncytial blastoderm. The promoters of these genes have been used in genetic pest-control systems to express transgenes that confer embryonic lethality. Such systems could be helpful in sterile insect technique applications to ensure that sterility (bi-sex embryonic lethality) or sexing (female-specific embryonic lethality) can be achieved during mass rearing. The activity of cellularization gene promoters during embryogenesis controls the timing and dose of the lethal gene product.ResultsHere, we report the isolation of the D. suzukii cellularization genes nullo, serendipity-α, bottleneck and slow-as-molasses from a laboratory strain. Conserved motifs were identified by comparing the encoded proteins with orthologs from other Drosophilids. Expression profiling confirmed that all four are zygotic genes that are strongly expressed at the early blastoderm stage. The 5′ flanking regions from these cellularization genes were isolated, incorporated into piggyBac vectors and compared in vitro for the promoter activities. The Dsnullo promoter showed the highest activity in the cell culture assays using D. melanogaster S2 cells.ConclusionsThe similarities in the gene coding and 5′ flanking sequence as well as in the expression pattern of the four cellularization genes between D. melanogaster and D. suzukii, suggest that conserved functions may be involved in both species. The high expression level at the early blastoderm stage of the four cellularization genes were confirmed, thus their promoters can be considered in embryonic lethality systems. While the Dsnullo promoter could be a suitable candidate, all reported promoters here are subject to further in vivo analyses before constructing potential pest control systems.

An early female lethal system of the New World screwworm, Cochliomyia hominivorax, for biotechnology-enhanced SIT

BackgroundThe New World Screwworm fly (NWS), Cochliomyia hominivorax, is an ectoparasite of warm-blooded animals and a major pest of livestock in parts of South America and the Caribbean where it remains endemic. In North and Central America it was eradicated using the Sterile Insect Technique (SIT). A control program is managed cooperatively between the governments of the United States and Panama to prevent the northward spread of NWS from infested countries in South America. This is accomplished by maintaining a permanent barrier through the release of millions of sterile male and female flies in the border between Panama and Colombia. Our research team demonstrated the utility of biotechnology-enhanced approaches for SIT by developing a male-only strain of the NWS. The strain carried a single component tetracycline repressible female lethal system where females died at late larval/pupal stages. The control program can be further improved by removing females during embryonic development as larval diet costs are significant.ResultsThe strains developed carry a two-component system consisting of the Lucilia sericata bottleneck gene promoter driving expression of the tTA gene and a tTA-regulated Lshid proapoptotic effector gene. Insertion of the sex-specifically spliced intron from the C. hominivorax transformer gene within the Lshid gene ensures that only females die when insects are reared in the absence of tetracycline. In several double homozygous two-component strains and in one “All-in-one” strain that had both components in a single construct, female lethality occurred at the embryonic and/or first instar larval stages when raised on diet without tetracycline. Laboratory evaluation for phenotypes that are relevant for mass rearing in a production facility revealed that most strains had fitness characteristics similar to the wild type J06 strain that is currently reared for release in the permanent barrier. Testing of an “All in one” strain under mass rearing conditions showed that the strain maintained the fitness characteristics observed in small-scale rearing.ConclusionsThe early female lethal strains described here could be selected by the NWS Control Program for testing at large scale in the production facility to enhance the efficiency of the NWS eradication program.

Building a transgenic sexing strain for genetic control of the Australian sheep blow fly Lucilia cuprina using two lethal effectors

BackgroundThe sterile insect technique (SIT) has been successfully used in many pest management programs worldwide. Some SIT programs release both sexes due to the lack of genetic sexing strains or efficient sex separation methods but sterile females are ineffective control agents. Transgenic sexing strains (TSS) using the tetracycline-off control system have been developed in a variety of insect pests, from which females die by either of two commonly used lethal effectors: overexpression of the transcription factor tetracycline transactivator (tTA) or ectopic expression of a proapoptotic gene, such as head involution defective (hid). The lethality from tTA overexpression is thought to be due to “transcriptional squelching”, while hid causes lethality by induction of apoptosis. This study aims to create and characterize a TSS of Lucilia cuprina, which is a major pest of sheep, by combining both lethal effectors in a single transgenic strain.ResultsHere a stable TSS of L. cuprina (DH6) that carries two lethal effectors was successfully generated, by crossing FL3#2 which carries a female-specific tTA overexpression cassette, with EF1#12 which carries a tTA-regulated LshidAla2 cassette. Females with one copy of the FL3#2 transgene are viable but up to 99.8% of homozygous females die at the pupal stage when raised on diet that lacks tetracycline. Additionally, the female lethality of FL3#2 was partially repressed by supplying tetracycline to the parental generation. With an additional LshidAla2 effector, the female lethality of DH6 is 100% dominant and cannot be repressed by maternal tetracycline. DH6 females die at the late-larval stage. Several fitness parameters important for mass rearing such as hatching rate, adult emergence and sex ratio were comparable to those of the wild type strain.ConclusionsCompared to the parental FL3#2 strain, the DH6 strain shows stronger female lethality and lethality occurs at an earlier stage of development. The combination of two tTA-dependent lethal effectors could improve strain stability under mass rearing and could reduce the risk of resistance in the field if fertile males are released. Our approach could be easily adapted for other pest species for an efficient, safe and sustainable genetic control program.

Precise single base substitution in the shibire gene by CRISPR/Cas9-mediated homology directed repair in Bactrocera tryoni

BackgroundPest eradication using the Sterile Insect Technique (SIT) involves high-density releases of sterilized males that mate with wild females and ultimately suppress the population. Sterilized females are not required for SIT and their removal or separation from males prior to release remains challenging. In order to develop genetic sexing strains (GSS), conditional traits such as temperature sensitive lethality are required.ResultsHere we introduce a known Drosophila melanogaster temperature sensitive embryonic lethal mutation into Bactrocera tryoni, a serious horticultural pest in Australia. A non-synonymous point mutation in the D. melanogaster gene shibire causes embryonic lethality at 29 °C and we successfully used CRISPR/Cas9 technology to recreate the orthologous shibire temperature sensitive-1 (shits1) mutation in B. tryoni. Genotypic analyses over three generations revealed that a high fitness cost was associated with the shits1 mutant allele and shits1 homozygotes were not viable at 21 °C, which is a more severe phenotype than that documented in D. melanogaster.ConclusionsWe have demonstrated the first successful use of CRISPR/Cas9 to introduce precise single base substitutions in an endogenous gene via homology-directed repair in an agricultural pest insect and this technology can be used to trial other conditional mutations for the ultimate aim of generating genetic sexing strains for SIT.

Irradiation induced inversions suppress recombination between the M locus and morphological markers in Aedes aegypti

BackgroundAedes aegypti is the primary vector of arthropod-borne viruses and one of the most widespread and invasive mosquito species. Due to the lack of efficient specific drugs or vaccination strategies, vector population control methods, such as the sterile insect technique, are receiving renewed interest. However, availability of a reliable genetic sexing strategy is crucial, since there is almost zero tolerance for accidentally released females. Development of genetic sexing strains through classical genetics is hindered by genetic recombination that is not suppressed in males as is the case in many Diptera. Isolation of naturally-occurring or irradiation-induced inversions can enhance the genetic stability of genetic sexing strains developed through genetically linking desirable phenotypes with the male determining region.ResultsFor the induction and isolation of inversions through irradiation, 200 male pupae of the ‘BRA’ wild type strain were irradiated at 30 Gy and 100 isomale lines were set up by crossing with homozygous ‘red-eye’ (re) mutant females. Recombination between re and the M locus and the white (w) gene (causing a recessive white eye phenotype when mutated) and the M locus was tested in 45 and 32 lines, respectively. One inversion (Inv35) reduced recombination between both re and the M locus, and wand the M locus, consistent with the presence of a rather extended inversion between the two morphological mutations, that includes the M locus. Another inversion (Inv5) reduced recombination only between w and the M locus. In search of naturally-occurring, recombination-suppressing inversions, homozygous females from the red eye and the white eye strains were crossed with seventeen and fourteen wild type strains collected worldwide, representing either recently colonized or long-established laboratory populations. Despite evidence of varying frequencies of recombination, no combination led to the elimination or substantial reduction of recombination.ConclusionInducing inversions through irradiation is a feasible strategy to isolate recombination suppressors either on the M or the m chromosome for Aedes aegypti. Such inversions can be incorporated in genetic sexing strains developed through classical genetics to enhance their genetic stability and support SIT or other approaches that aim to population suppression through male-delivered sterility.

Transcriptome analysis of Anastrepha fraterculus sp. 1 males, females, and embryos: insights into development, courtship, and reproduction

BackgroundAnastrepha fraterculus sp. 1 is considered a quarantine pest in several American countries. Since chemical control applied in an integrated pest management program is the only strategy utilized against this pest, the development of pesticide-free methods, such as the Sterile Insect Technique, is being considered. The search for genes involved in sex-determination and differentiation, and in metabolic pathways associated with communication and mating behaviour, contributes with key information to the development of genetic control strategies. The aims of this work were to perform a comprehensive analysis of A. fraterculus sp. 1 transcriptome and to obtain an initial evaluation of genes associated with main metabolic pathways by the expression analysis of specific transcripts identified in embryos and adults.ResultsSexually mature adults of both sexes and 72 h embryos were considered for transcriptome analysis. The de novo transcriptome assembly was fairly complete (62.9% complete BUSCO orthologs detected) with a total of 86,925 transcripts assembled and 28,756 GO annotated sequences. Paired-comparisons between libraries showed 319 transcripts differently expressed between embryos and females, 1242 between embryos and males, and 464 between sexes. Using this information and genes searches based on published studies from other tephritid species, we evaluated a set of transcripts involved in development, courtship and metabolic pathways. The qPCR analysis evidenced that the early genes serendipity alpha and transformer-2 displayed similar expression levels in the analyzed stages, while heat shock protein 27 is over-expressed in embryos and females in comparison to males. The expression of genes associated with courtship (takeout-like, odorant-binding protein 50a1) differed between males and females, independently of their reproductive status (virgin vs mated individuals). Genes associated with metabolic pathways (maltase 2-like, androgen-induced gene 1) showed differential expression between embryos and adults. Furthermore, 14,262 microsatellite motifs were identified, with 11,208 transcripts containing at least one simple sequence repeat, including 48% of di/trinucleotide motifs.ConclusionOur results significantly expand the available gene space of A. fraterculus sp. 1, contributing with a fairly complete transcript database of embryos and adults. The expression analysis of the selected candidate genes, along with a set of microsatellite markers, provides a valuable resource for further genetic characterization of A. fraterculus sp. 1 and supports the development of specific genetic control strategies.

Transcribed sex-specific markers on the Y chromosome of the oriental fruit fly, Bactrocera dorsalis

BackgroundThe Oriental fruit fly, Bactrocera dorsalis, is a highly polyphagous invasive species with a high reproductive potential. In many tropical and subtropical parts of the world it ranks as one of the major pests of fruits and vegetables. Due to its economic importance, genetic, cytogenetic, genomic and biotechnological approaches have been applied to understand its biology and to implement the Sterile Insect Technique, currently a part of area-wide control programmes against this fly. Its chromosome complement includes five pairs of autosomes and the sex chromosomes. The X and Y sex chromosomes are heteromorphic and the highly heterochromatic and degenerate Y harbours the male factor BdMoY. The characterization of the Y chromosome in this fly apart from elucidating its role as primary sex determination system, it is also of crucial importance to understand its role in male biology. The repetitive nature of the Y chromosome makes it challenging to sequence and characterise.ResultsUsing Representational Difference Analysis, fluorescent in situ hybridisation on mitotic chromosomes and in silico genome resources, we show that the B. dorsalis Y chromosome harbours transcribed sequences of gyf, (typo-gyf) a homologue of the Drosophila melanogaster Gigyf gene, and of a non-LTR retrotransposon R1. Similar sequences are also transcribed on the X chromosome. Paralogues of the Gigyf gene are also present on the Y and X chromosomes of the related species B. tryoni. Another identified Y-specific repetitive sequence linked to BdMoY appears to be specific to B. dorsalis.ConclusionsOur random scan of the Y chromosome provides a broad picture of its general composition and represents a starting point for further applicative and evolutionary studies. The identified repetitive sequences can provide a useful Y-marking system for molecular karyotyping of single embryos. Having a robust diagnostic marker associated with BdMoY will facilitate studies on how BdMoY regulates the male sex determination cascade during the embryonic sex-determination window. The Y chromosome, despite its high degeneracy and heterochromatic nature, harbours transcribed sequences of typo-gyf that may maintain their important function in post-transcriptional mRNA regulation. That transcribed paralogous copies of Gigyf are present also on the X and that this genomic distribution is maintained also in B. tryoni raises questions on the evolution of sex chromosomes in Bactrocera and other tephritids.

Genetic stability, genetic variation, and fitness performance of the genetic sexing Salaya1 strain for Bactrocera dorsalis, under long-term mass rearing conditions

BackgroundA genetic sexing strain (GSS) is an essential component for pest control using the sterile insect technique (SIT). A GSS is developed using a combination of Y-autosome translocation and a selectable marker such as pupal color, resulting in heterozygous males and homozygous females that possess wild-type brown pupae (wp+) and mutant white pupae (wp) alleles, respectively. The genetic sexing Salaya1 strain developed for Bactrocera dorsalis was evaluated using a clean stream and scaled-up for subsequent production lines (e.g., initiation, injection, and release). Colony management under small- and large-scale conditions for long-term rearing may affect the sexing system, genetic background, and fitness performance of the strain. Routine monitoring was applied to study genetic stability, genetic variation, and male mating competitiveness.ResultsThe percentage of recombinants was significantly different between males (wp) and females (wp+), ranging between 0.21–0.43% and 0.01–0.04%, respectively. Using 106 bands from six ISSR markers, the genetic backgrounds of two generations (F40 and F108) of the clean stream were found to be almost identical (0.960), and between those two generations and the wild population, the similarities were 0.840 and 0.800, respectively. In addition, the sterile males performed well in competitive mating with fertile females (Relative Sterility Index = 0.67 ± 0.13). The rates of fliers calculated from both clean and release streams were higher than 0.95. Regarding the fitness of the Salaya1 strain, the fertility and pupal recovery were similar in all production lines. The sex ratio (Male/Female) distortion was also recorded.ConclusionsThe Salaya1 strain reared at the mass-rearing facility retained its genetic stability, genetic variation, behavior (e.g., competitive mating and flight ability), and traits related to fitness for at least 10 consecutive generations. The filter rearing system is effective at minimising the selection pressure while maintaining the genetic background and fitness performances of the clean stream. These characteristics were stable throughout the production lines. In addition, the production efficiency is comparable among the different production lines and other similar types of GSSs.

Genetic structure and symbiotic profile of worldwide natural populations of the Mediterranean fruit fly, Ceratitis capitata

BackgroundThe Mediterranean fruit fly, Ceratitis capitata, is a cosmopolitan agricultural pest of worldwide economic importance and a model for the development of the Sterile Insect Technique (SIT) for fruit flies of the Tephritidae family (Diptera). SIT relies on the effective mating of laboratory-reared strains and natural populations, and therefore requires an efficient mass-rearing system that will allow for the production of high-quality males. Adaptation of wild flies to an artificial laboratory environment can be accompanied by negative effects on several life history traits through changes in their genetic diversity and symbiotic communities. Such changes may lead to reduced biological quality and mating competitiveness in respect to the wild populations. Profiling wild populations can help understand, and maybe reverse, deleterious effects accompanying laboratory domestication thus providing insects that can efficiently and effectively support SIT application.ResultsIn the present study, we analyzed both the genetic structure and gut symbiotic communities of natural medfly populations of worldwide distribution, including Europe, Africa, Australia, and the Americas. The genetic structure of 408 individuals from 15 distinct populations was analyzed with a set of commonly used microsatellite markers. The symbiotic communities of a subset of 265 individuals from 11 populations were analyzed using the 16S rRNA gene-based amplicon sequencing of single individuals (adults). Genetic differentiation was detected among geographically distant populations while adults originated from neighboring areas were genetically closer. Alpha and beta diversity of bacterial communities pointed to an overall reduced symbiotic diversity and the influence of the geographic location on the bacterial profile.ConclusionsOur analysis revealed differences both in the genetic profile and the structure of gut symbiotic communities of medfly natural populations. The genetic analysis expanded our knowledge to populations not analyzed before and our results were in accordance with the existing scenarios regarding this species expansion and colonization pathways. At the same time, the bacterial communities from different natural medfly populations have been characterized, thus broadening our knowledge on the microbiota of the species across its range. Genetic and symbiotic differences between natural and laboratory populations must be considered when designing AW-IPM approaches with a SIT component, since they may impact mating compatibility and mating competitiveness of the laboratory-reared males. In parallel, enrichment from wild populations and/or symbiotic supplementation could increase rearing productivity, biological quality, and mating competitiveness of SIT-important laboratory strains.

Effect of Glomus fasciculatum on Biochemical Composition of Catharanthus roseus L.

Catharanthus roseus was selected for the present investigation because it is the most important medicinal plant in India. It consists of abortifacient effect, acid phosphatase inhibition, acid phosphatase stimulation anti-ascariasis activity, antibacterial activity, antidiuretic activity, antihyperglycemic activity, antihypertensive activity, anti-inflammatory activity, antimitotic activity, antispasmodic anti-viral activity, cardiotonic activity, cytotoxic activity, hyperglycemic activity, hypotensive activity, insect sterility induction activity, leucopenic activity, uterine stimulant effect, as well as, weight loss activity, etc. The purpose of this study was to observe the effect of mycorrhizae, like Glomus fasciculatum on C. roseus and its different biochemical composition. The leaves of Catharanthus showed good antioxidant activity (82.17%) and UV-visible spectra show a growth factor of plant. These observations are suggested that the use of mycorrhiza, like G. fasciculatum for the growth of the plant, is useful and also shows a positive effect on the biochemical composition of plant C. roseus.

Combined Effects of Mating Disruption, Insecticides, and the Sterile Insect Technique on Cydia pomonella in New Zealand.

Simple SummaryCodling moth is a major pest of apples, and was accidentally introduced into New Zealand over 150 years ago. Many countries that New Zealand exports apples to do not have codling moth present and they want to keep it out. Therefore, apple growers must heavily control codling moth populations on their orchards. Currently, the main control tactics are insecticide applications and mating disruption, which uses the moth’s own sex pheromone to make the males unable to find females to mate. We aimed to supplement these tactics with the sterile insect technique (SIT) to further suppress the codling moth on orchards. SIT involves mass rearing and sterilizing codling moth and then releasing them onto orchards where they mate with wild insects resulting in no offspring. We released sterile insects onto seven orchards using unmanned aerial vehicles and ground releases. Six years of the program saw significant drops (90–99%) in wild moth populations. The SIT is an excellent tactic for reducing moth populations in export apple orchards.Codling moth was introduced into New Zealand, and remains a critical pest for the apple industry. Apples exported to some markets require strict phytosanitary measures to eliminate the risk of larval infestation. Mating disruption and insecticide applications are the principal means of suppression in New Zealand. We tested the potential for the sterile insect technique (SIT) to supplement these measures to achieve local eradication or suppression of this pest. SIT was trialed in an isolated group of six integrated fruit production (IFP) orchards and one organic orchard (total 391 ha), using sterilized insects imported from Canada, with release by unmanned aerial vehicle and from the ground. Eradication was not achieved across the region, but a very high level of codling moth suppression was achieved at individual orchards after the introduction of sterile moths in combination with mating disruption and larvicides. After six years of releases, catches of wild codling moths at three IFP orchards (224 ha) were 90–99% lower than in 2013–2014, the year before releases began. Catches at three other IFP orchards (129 ha) decreased by 67–97% from the year before releases began (2015–2016), from lower initial levels. At a certified organic orchard with a higher initial population under only organic larvicides and mating disruption, by 2019–2020, there was an 81% reduction in wild moths capture from 2016–2017, the year before releases began.

Physiological and molecular correlates of the screwworm fly attraction to wound and animal odors

The screwworm fly, Cochliomyia hominivorax (Coquerel), was successfully eradicated from the United States by the sterile insect technique (SIT). However, recent detection of these flies in the Florida Keys, and increased risk of introductions to the other areas warrant novel tools for management of the flies. Surveillance, a key component of screwworm control programs, utilizes traps baited with rotting liver or a blend of synthetic chemicals such as swormlure-4. In this work, we evaluated the olfactory physiology of the screwworm fly and compared it with the non-obligate ectoparasitic secondary screwworm flies, C. macellaria, that invade necrotic wound and feed on dead tissue. These two species occur in geographically overlapping regions. C. macellaria, along with other blowflies such as the exotic C. megacephala, greatly outnumber C. hominivorax in the existing monitoring traps. Olfactory responses to swormlure-4 constituents between sex and mating status (mated vs unmated) in both species were recorded and compared. Overall, responses measured by the antennograms offered insights into the comparative olfactory physiology of the two fly species. We also present detailed analyses of the antennal transcriptome by RNA-Sequencing that reveal significant differences between male and female screwworm flies. The differential expression patterns were confirmed by quantitative PCR. Taken together, this integrated study provides insights into the physiological and molecular correlates of the screwworm’s attraction to wounds, and identifies molecular targets that will aid in the development of odorant-based fly management strategies.

Disruption of duplicated yellow genes in Bactrocera tryoni modifies pigmentation colouration and impacts behaviour

Irradiated Queensland fruit flies (Bactrocera tryoni) used in Sterile Insect Technique (SIT) programmes are marked with fluorescent dyes to distinguish them from wild flies when recaptured in monitoring traps. However, coating sterile pupae with powdered dyes can reduce emergence rates and fly quality and can sometimes produce insufficiently certain discrimination through inadequate coating or because the dye is transferred to wild flies through contact. Here we created a phenotypically distinct B. tryoni strain that lacks typical melanisation patterns through CRISPR/Cas9-mediated mutagenesis of tandemly duplicated yellow-y genes and then assessed effects of this visible trait on fly performance. Recessive mutations are only required in one of these copies to restrict melanisation and generate a phenotype clearly distinguished from wild type. The yellow strain showed significant declines in eclosion rates and in the percentage of fliers directly after emergence. Locomotor activity was greater in the yellow strain, and these mutations did not generally affect mating probability, copula latency, or copula duration. The longevity of yellow flies was approximately 10 days shorter than wild-type flies in both sexes. Overall, replacing dyes with yellow body marker for SIT can simplify production, eliminate a step that is known to reduce fly quality, remove potentially hazardous dyes from production, enable accurate discrimination from wild flies, and improve cost-effectiveness; however, direct comparisons of the decrements in performance associated with dyes on mass-reared wild-type flies and disruption of yellow-y genes are now required to determine the relative suitability of these marking methods for B. tryoni SIT.