Genomic targeting by recombinase-mediated cassette exchange in the spotted wing drosophila, Drosophila suzukii.

Drosophila suzukii is a significant pest of stone and small fruits. The genome of this species has been sequenced and manipulated by transposon-mediated transformation and CRISPR/Cas9 gene editing. These technologies open a variety of possibilities for functional genomics and genetic modifications that might improve biologically based population control strategies. Both of these approaches, however, would benefit from genome targeting that would avoid position effects and insertional mutations associated with random transposon vector insertions, and the limited DNA fragment insertion size allowed by gene editing. Here, we describe an efficient recombinase-mediated cassette exchange (RMCE) system for D. suzukii in which heterospecific lox recombination sites were integrated into the genome by transposon-mediated transformation and subsequently targeted for double recombination by a donor vector in the presence of Cre recombinase. Three loxN/lox2272 landing site lines have previously been created in D. suzukii, and quantitative PCR determined that polyubiquitin-regulated enhanced green fluorescent protein expression is least susceptible to position effect suppression in the 443_M26m1 line. We presume that RMCE target sites may also be inserted more specifically into the genome by homology-directed repair gene editing, thereby avoiding position effects and mutations, while eliminating restrictions on the size of donor constructs for subsequent insertion.

Tetracycline-suppressible female lethality and sterility in the Mexican fruit fly, Anastrepha ludens.

The sterile insect technique (SIT) involves the mass release of sterile males to suppress insect pest populations. SIT has been improved for larval pests by the development of strains for female-specific tetracycline-suppressible (Tet-off) embryonic lethal systems for male-only populations. Here we describe the extension of this approach to the Mexican fruit fly, Anastrepha ludens, using a Tet-off driver construct with the Tet-transactivator (tTA) under embryo-specific Anastrepha suspensa serendipity α (As-sry-α) promoter regulation. In the absence of tetracycline, tTA acts upon a Tet-response element linked to the pro-apoptotic cell death gene lethal effector, head involuation defective (hid), from A. ludens (Alhid(Ala2) ) that contains a sex-specific intron splicing cassette, resulting in female-specific expression of the lethal effector. Parental adults double-homozygous for the driver/effector vectors were expected to yield male-only progeny when reared on Tet-free diet, but a complete lack of oviposited eggs resulted for each of the three strains tested. Ovary dissection revealed nonvitellogenic oocytes in all strains that was reversible by feeding females tetracycline for 5 days after eclosion, resulting in male-only adults in one strain. Presumably the sry-α promoter exhibits prezygotic maternal expression as well as zygotic embryonic expression in A. ludens, resulting in a Tet-off sterility effect in addition to female-specific lethality.

Not all GMOs are crop plants: non-plant GMO applications in agriculture.

Since tools of modern biotechnology have become available, the most commonly applied and often discussed genetically modified organisms are genetically modified crop plants, although genetic engineering is also being used successfully in organisms other than plants, including bacteria, fungi, insects, and viruses. Many of these organisms, as with crop plants, are being engineered for applications in agriculture, to control plant insect pests or diseases. This paper reviews the genetically modified non-plant organisms that have been the subject of permit approvals for environmental release by the United States Department of Agriculture/Animal and Plant Health Inspection Service since the US began regulating genetically modified organisms. This is an indication of the breadth and progress of research in the area of non-plant genetically modified organisms. This review includes three examples of promising research on non-plant genetically modified organisms for application in agriculture: (1) insects for insect pest control using improved vector systems; (2) fungal pathogens of insects to control insect pests; and (3) virus for use as transient-expression vectors for disease control in plants.

Germ-line transformation of the Queensland fruit fly, Bactrocera tryoni, using a piggyBac vector in the presence of endogenous piggyBac elements.

We report the heritable germ-line transformation of the Queensland fruit fly, Bactrocera tryoni, using a piggyBac vector marked with either the fluorescent protein DsRed or EGFP. A transformation frequency of 5-10% was obtained. Inheritance of the transgenes has remained stable over more than 15 generations despite the presence of endogenous piggyBac sequences in the B. tryoni genome. The sequence of insertion sites shows the usual canonical pattern of piggyBac integraton into TTAA target sites. An investigation of endogenous piggyBac elements in the B. tryoni genome reveals the presence of sequences almost identical to those reported recently for the B. dorsalis complex of fruit flies and two noctuid moths, suggesting a common origin of piggyBac sequences in these species. The availability of transformation protocols for B. tryoni has the potential to deliver improvements in the performance of the Sterile Insect Technique for this pest species.

Characterization of the proteasomebeta2 subunit gene and its mutant allele in the tephritid fruit fly pest, Anastrepha suspensa.

In Drosophila melanogaster the beta2 proteasome subunit gene, Prosbeta2, was first identified as a dominant temperature sensitive mutant, DTS-7, that causes pupal lethality at 29 degrees C but allows survival to adulthood at 25 degrees C. To explore the use of proteasome mutations for a conditional lethal system in insect pests, we identified and isolated the beta2 subunit gene of the 20S proteasome from the Caribbean fruit fly, Anastrepha suspensa. The caribfly ortholog AsProsbeta2 was isolated from pupal cDNA by 5' and 3' RACE. The AsProsbeta2 protein has high amino acid sequence similarity to predicted insect Prosbeta2 subunits and homologs from yeast and mammals, and it contains the well conserved amino acids that confer catalytic activity and substrate specificity. AsProsbeta2 is a single copy gene and its RNA accumulates throughout all developmental stages of the caribfly. For functional studies a point mutation, analogous to the Prosbeta2(1) mutation in D. melanogaster, was introduced into AsProsbeta2 to create an aberrant protein with a Gly170Arg substitution. Consistent with the DTS-7 mutation, transgenic insects carrying the mutant allele undergo normal metamorphosis at the permissive temperature (25 degrees C) but at the non-permissive temperature (29 degrees C) they exhibit effective pupal lethality. This is the first report of a functional characterization of a Prosbeta2 cognate based on the creation of a dominant temperature-sensitive mutation. This type of temperature-dependent lethality could be used for biological control, where transgenic insects are reared to adulthood at 25 degrees C or lower and then released into the field where ambient temperatures averaging 29 degrees C or greater cause lethality in their progeny.

Development and utilization of transgenic New World screwworm, Cochliomyia hominivorax.

The New World screwworm (NWS), Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae), was the first insect to be effectively controlled using the sterile insect technique (SIT). Recent efforts to improve SIT control of this species have centred on the development of genetically transformed strains using the piggyBac transposon vector system. Eight transgenic strains were produced incorporating an enhanced green fluorescent protein (EGFP) marker gene under polyubiquitin regulation that has the potential for use as a genetic marking system for released males. The transgenic strains were genetically and phenotypically characterized, including for life fitness parameters and mating competitiveness. These characteristics were unique for each strain and thus some strains were deemed suitable for incorporation into SIT eradication programmes. The strain with the best attributes is designated 'CLAY'. Four of the strains, including CLAY, have been successfully cryopreserved so that their original characteristics should be unchanged when further evaluation is required. With the demonstration of efficient germ-line transformation in NWS, allowing production of fit and competitive transformants, it is now possible to consider further transgenic strain development to improve SIT that are currently being tested in other dipteran species. This includes strains that allow genetic marking with fluorescent proteins, genetic sexing by female lethality, male-specific fluorescent sorting and male sterility by testis-specific lethality. The SIT may also be improved upon by new strategies resulting in lethality of offspring of released insects using conditional lethal systems based upon temperature-dependent or dietary tetracycline regulation of lethal gene expression. Both the creation of new NWS transgenic strains and the ecological safety of their release will be enhanced by new vector systems that allow specific genomic targeting of vector constructs and their subsequent immobilization, ensuring transgene and strain stability.

Highly similar piggyBac elements in Bactrocera that share a common lineage with elements in noctuid moths.

The piggyBac IFP2 transposable element, originally discovered in a Trichoplusia ni cell line, also exists as nearly identical elements in other noctuid lepidopterans, and in several species of the tephritid genus Bactrocera. To further define the distribution of piggyBacs in Bactrocera, and compare their relationship to sequences found in Lepidoptera, a survey by PCR amplification was performed in a range of Bactrocera species. Highly similar piggyBac sequences were found in all B. dorsalis complex species tested, as well as in species in the B. zonata and B. frauenfeldi complexes. All nucleotide sequences had > 94% identity to corresponding sequences in the T. ni IFP2 element, and > 88% identity among the sequences. Conserved primers did not amplify any distantly related sequences that have been found by computational searches in a wider range of insect and non-insect species. Notably, 55 nucleotide substitutions relative to IFP2 were common to all the Bactrocera sequences, 44 of which exist in piggyBacs previously sequenced from moths, with 17 resulting in amino acid substitutions. These piggyBac elements, that apparently traversed orders by horizontal transfer, probably arose from a lineage separate from IFP2 and the other known elements in T. ni. Implications for the presence of nearly identical piggyBacs, in widely distributed insects, to the applied use of piggyBac vectors are discussed.

Adenocarcinoma of the glandular stomach following 3-methylcholanthrene, N-nitrosodiethylamine, or N-nitrosodimethylamine feeding in carcinogen-susceptible inbred Syrian.

Invasive adenocarcinoma of the glandular stomach was induced in 3 BIO 87.20 inbred Syrian hamsters by the administration of 1 ppm N-nitrosodiethylamine or N-nitrosodimethylamine in the drinking water, and in 2 BIO 15.16 and 5 BIO 72.29 inbred Syrian hamsters by gavage of 3-methylcholanthrene. In contrast, many other similarly treated inbred Syrian hamster lines did not develop carcinoma of the glandular stomach. The glandular stomachs of rats and mice are resistant to polycyclic hydrocarcon carcinogenesis; however, the glandular stomachs of certain inbred Syrian hamsters develop adenocarcinoma after polycyclic hydrocarbon gavage. This work demonstrated the importance of genetic factors.

The hobo transposable element excises and has related elements in tephritid species.

Function of the Drosophila melanogaster hobo transposon in tephritid species was tested in transient embryonic excision assays. Wild-type and mutant strains of Anastrepha suspensa, Bactrocera dorsalis, B. cucurbitae, Ceratitis capitata, and Toxotrypana curvicauda all supported hobo excision or deletion both in the presence and absence of co-injected hobo transposase, indicating a permissive state for hobo mobility and the existence of endogenous systems capable of mobilizing hobo. In several strains hobo helper reduced excision. Excision depended on hobo sequences in the indicator plasmid, though almost all excisions were imprecise and the mobilizing systems appear mechanistically different from hobo. hobo-related sequences were identified in all species except T. curvicauda. Parsimony analysis yielded a subgroup including the B. cucurbitae and C. capitata sequences along with hobo and Hermes, and a separate, more divergent subgroup including the A. suspensa and B. dorsalis sequences. All of the sequences exist as multiple genomic elements, and a deleted form of the B. cucurbitae element exists in B. dorsalis. The hobo-related sequences are probably members of the hAT transposon family with some evolving from distant ancestor elements, while others may have originated from more recent horizontal transfers.

A new hobo, Ac, Tam3 transposable element, hopper, from Bactrocera dorsalis is distantly related to hobo and Ac.

A new transposable element from the hobo, Ac, Tam3 transposon family was isolated as a genomic clone from the oriental fruit fly, Bactrocera dorsalis. It is approximately 3.1 kb in length with 19-bp inverted terminal repeat sequences having a single mismatch. Though sharing several amino acid sequence identities with other hAT elements, it is distantly related to both hobo and Ac. Among hAT elements thus far described in insects, it is apparently the most distantly related to hobo.

Taurine-induced synaptic potentiation and the late phase of long-term potentiation are related mechanistically.

The application of taurine (2-aminoethanesulfonic acid) induces a long-lasting increase of synaptic efficacy and axon excitability (LLP-TAU) in rat hippocampal CA1 area. After taurine withdrawal, LLP-TAU lasted at least 3 h. This fact prompted us to assess whether the mechanisms involved in the maintenance of this particular potentiation were similar to those implicated in the late phase of long-term potentiation (L-LTP). In the presence of KN-62, an inhibitor of calcium/calmodulin-dependent protein kinase, taurine perfusion (10 mM, 30 min) did not affect the induction of LLP-TAU. However, LLP-TAU maintenance was completely suppressed by KT5720, an inhibitor of the cAMP-dependent protein kinase (PKA). Moreover, the late phase of LLP-TAU was blocked by inhibiting protein synthesis with anisomycin. In addition, taurine perfusion increased the phosphorylation of cAMP response element-binding protein (CREB), although did not affect cAMP levels. These features of LLP-TAU do not appear to be caused by the activation of D1/D5 dopamine receptors, as taurine also induced synaptic potentiation in the presence of SCH23390, an antagonist of this type of receptors. Finally, the late phase of both L-LTP and LLP-TAU occluded mutually. These results suggest that taurine triggers the sequence of some of the molecular events involved in the induction of L-LTP.

Polyubiquitin-regulated DsRed marker for transgenic insects.

Genetic transformation of most insect systems requires dominant-acting markers that do not depend on reverting a mutant phenotype in a host strain, andfor this purpose GFP has proven to be useful in several insect orders. However, detection of multiple transgenes and reporters for gene expression requires the development of new visible markers that can be unambiguously detected when co-expressed with GFP The DsRed fluorescentprotein has spectral characteristics that are most distinct from GFP and GFP variants, and we have explored the use of DsRed as a selectable marker for piggyBac transformation in Drosophila melanogaster and its use as a reporter when co-expressed with GFP. Transformants marked with polyubiquitin-regulated DsRed1 were detected throughout development at a relatively high frequency, and they exhibited brighter fluorescence than transformants marked with EGFP. The use of a Texas Red filter set eliminated detection of EGFP fluorescence and autofluorescence, and DsRed expressedfrom a reporter construct could be unambiguously detected when co-expressed with EGFP DsRed should prove to be a highly efficient marker system for the selection of transformant insects and as a reporter in gene expression studies.

Ecdysteroid receptors in Drosophila melanogaster adult females.

Ecdysteroid receptors were identified and partially characterized from total cell extracts of whole animals and dissected tissues from Drosophila melanogaster adult females. Binding studies indicated the presence of two ecdysteroid binding components having high affinity and specificity consistent with receptors previously identified in embryos and larvae. The highest affinity binding component in 3- to 4-day females had a dissociation constant of 9.2 x 10(-10) M and a maximal binding concentration of approximately 90 pmol/g protein, with a lower affinity component having a dissociation constant of 2.94 X 10(-9) M. Receptors at similar concentrations were also observed in abdominal walls containing adult fat body, with relatively lower receptor levels observed in ovaries. These results indicate that the observed ecdysteroid hormone concentrations in adult females can account for a physiological stimulatory effect on yolk protein synthesis in adult fat body.

A current perspective on insect gene transformation.

The genetic transformation of non-drosophilid insects is now possible with several systems, with germ-line transformation reported in published and unpublished accounts for about 12 species using four different transposon vectors. For some of these species, transformation can now be considered routine. Other vector systems include viruses and bacterial symbionts that have demonstrated utility in species and applications requiring transient expression, and for some, the potential exists for genomic integration. Many of these findings are quite recent, presenting a dramatic turning point in our ability to study and manipulate agriculturally and medically important insects. This review discusses these findings from the perspective of all the contributions that has made this technology a reality, the research that has yet to be done for its safe and efficient use in a broader range of species, and an overview of the available methodology to effectively utilize these systems.

Transformation of the Caribbean fruit fly, Anastrepha suspensa, with a piggyBac vector marked with polyubiquitin-regulated GFP.

Germ-line transformation was achieved in the Caribbean fruit fly, Anastrepha suspensa, using a piggyBac vector marked with an enhanced green fluorescent protein gene regulated by the Drosophila melanogaster polyubiquitin promoter. Four transgenic G(0) lines were selected exhibiting unambiguous GFP expression. Southern hybridization indicated the presence of one to four integrations in each of the transgenic lines with two integrations verified as piggyBac-mediated by sequencing their insertion sites. Fluorescence was detectable throughout development, and in adults was most intense from the thoracic flight muscle. Although adult cuticle quenched fluorescence, GFP was routinely detectable in the thorax. A quantitative spectrofluorometric assay was developed for GFP fluorescence that indicated differing levels of fluorescence among the transgenic lines, suggesting some level of position effect variegation/suppression. These results are encouraging for the use of this marker system in insect species not amenable to mutation-based visible markers. Together with the piggyBac vector, a transformation system is presented that has the potential to be universally applicable in insect species.

Evaluation of internal alpha-particle radiation exposure and subsequent fertility among a cohort of women formerly employed in the radium dial industry.

This study examined the effect of internal exposure to alpha-particle radiation on subsequent fertility among women employed in the radium dial industry prior to 1930, when appreciable amounts of radium were often ingested through the practice of pointing the paint brush with the lips. The analysis was limited to women for whom a radium body burden measurement had been obtained and who were married prior to age 45 (n = 603). Internal radiation dose to the ovary was calculated based on initial intakes of radium-226 and radium-228, average ovarian mass, number and energy of alpha particles emitted, fraction of energy absorbed within the ovary, effective retention integrals and estimated photon irradiation. Time between marriage and pregnancy, number of pregnancies and number of live births served as surrogates for fertility. Radiation appeared to have no effect on fertility at estimated cumulative ovarian dose equivalents below 5 Sv; above this dose, however, statistically significant declines in both number of pregnancies and live births were observed. These trends persisted after multivariable adjustment for potential confounding variables and after exclusion of subjects contributing a potential classification or selection bias to the study. Additionally, the high-dose group experienced fewer live births than would have been expected based on population rates. There were no differences in time to first pregnancy between high- and low-dose groups. These results are consistent with earlier studies of gamma-ray exposures and suggest that exposure to high doses of radiation from internally deposited radium reduces fertility rather than inducing sterility.

Preterm delivery and perinatal death among black and white infants in a Chicago-area perinatal registry.

OBJECTIVE: To explore associations between race, preterm delivery, etiologic classification of preterm delivery, and perinatal mortality. METHODS: The study population consisted of 13,010 black and 19,007 white mother-infant pairs delivered at Chicago-area hospitals in 1988-1989 categorized as term or preterm births. Preterm births were further divided by severity and etiology. Black-white differences in perinatal mortality within groups were calculated and adjusted for birth weight and other potential confounding variables. RESULTS: Black women were nearly twice as likely as whites to experience preterm (before 37 weeks' gestation) and very preterm (before 32 weeks' gestation) delivery associated with premature rupture of membranes (PROM) or classified as idiopathic. Although black infants were also found to have twice the perinatal mortality risk of white infants (relative risk [RR] 2.1, 95% confidence interval [CI] 1.7-2.5), the overall preterm perinatal mortality rates did not differ between black and white women (RR 1.0, 95% CI 0.8-1.2). However, among preterm births, perinatal mortality was not uniform within categories of medical etiology. The mortality risk was the same for black and white infants born preterm following polyhydramnios or placental complications (RR 1.1, 95% CI 0.6-1.9), the same for black and white infants born preterm after labor induction (RR 1.1, 95% CI 0.6-1.9), and higher for black infants classified as idiopathic preterm deliveries (RR 1.6, 95% CI 1.1-2.3). In contrast, mortality rates tended to be lower for black infants born preterm following PROM-amnionitis (RR 0.8, 95% CI 0.5-1.2). The idiopathic disparity was explained by a differential birth weight distribution (adjusted RR 1.1, 95% CI 0.7-1.9); however, the apparent survival benefit among black infants born preterm following PROM increased even further after adjustment for birth weight (adjusted RR 0.4, 95% CI 0.2-0.7). CONCLUSION: Black infants born preterm after PROM appear to have a survival advantage compared with their white counterparts, an effect not observed within other etiologic categories of preterm delivery.

A case-control study of 1253 twin pregnancies from a 1982-1987 perinatal data base.

In one regional perinatal network between 1982-1987, 101,506 women delivered infants greater than 500 g, of which 1253 were twin pregnancies (1.2%). This latter group was compared statistically with a 5% random sample of the singletons (N = 5119). The results showed that the women with twin pregnancies were slightly older, had a higher parity, gained more weight during the gestation, and had a heavier body weight at delivery. Twin pregnancies were complicated by increases in hypertension (odds ratio 2.5; 95% confidence interval 2.1-3.1), abruption (odds ratio 3.0; 95% confidence interval 1.9-4.7), and anemia (odds ratio 2.4; 95% confidence interval 1.9-3.0). There was no increased risk of pyelonephritis, placenta previa, or diabetes mellitus in mothers with twins. The twin pregnancies delivered earlier and the infants were smaller, had lower Apgar scores, and were at increased risk for congenital anomalies. Fetal and neonatal mortality rates were significantly increased in the twin infants; the perinatal mortality rates for twin A and twin B were 48.8 and 64.1, respectively, compared with 10.4 per 1000 births for the singleton controls. When the twin infants A and B were of similar weight, they had a similar perinatal mortality (odds ratio 1.0; 95% confidence interval 0.6-1.8). For infants less than 2500 g, twins A and B had lower fetal and neonatal mortality rates than did singletons, but twins heavier than 2500 g were at increased risk of perinatal death.

Roles for state-level local health liaison officials in local public health surveillance and capacity building.

In 27 state health agencies, local health liaison units or officials (LHLOs) are formally assigned responsibility for fostering a close working relationship between the state health agency and local health departments (LHDs). Yet in most other states, other agency staff carry out these responsibilities informally. Even where formal LHLOs exist, the assigned functions and specific activities vary, with little consistency across states other than serving as a potential or, in most cases, a real and vital linkage between LHDs and the state agency. This linkage places the LHLO in a unique position to have extensive knowledge, data, and information on LHD activities and to play an important role in assessing and improving local public health practice. This report examines aspects of the LHLO-LHD relationship in terms of potential LHLO roles in LHD practice surveillance and capacity building. Surveys of LHLOs and LHDs indicate that both support the development of surveillance tools to measure LHD effectiveness as a means to enhance capacity building efforts. Although LHLOs may not know or have the specific information immediately available to assess local public health practice for LHDs in their state, they report being able to obtain the information if necessary and with sufficient time. Further, LHDs are willing to share information concerning local public health practice with their state health department, particularly with their state LHLO. These findings suggest that LHLOs could be extensively involved in surveillance strategies beyond merely collecting and aggregating information provided by LHDs.

A strategy for measuring local public health practice.

The national health objectives for the year 2000 call for 90% of the population to be served by a local health department (LHD) that is effectively addressing the core functions of public health. Achieving this objective requires approved definitions for effectiveness as well as a system for ascertainment. In 1990 when this objective was established, no baseline data were available, and no accepted methods of measuring health department effectiveness were in use. Our approach to the development of a surveillance system to measure the effectiveness of LHDs has been to translate the three core public health functions characterized by the Institute of Medicine and the 10 practices delineated by the Centers for Disease Control and Prevention (CDC) and the national public health practice organizations into practice performance measures that could be judged as met or not met at the level of a jurisdiction served by a LHD. As part of our effort to develop a surveillance system to measure local public health practice, performance measures that characterize the 10 public health practices and their related core functions were developed and field-tested with state local health liaison officials and local health departments over a two-year period. Obtaining input from these sources is essential to establishing their validity and is a critical aspect of building nationwide consensus for appropriate measures of effective local public health practice. The results of these efforts led to the establishment of a proposed surveillance instrument comprising 10 performance standards and 29 associated indicators. We describe two approaches to its use.