CRISPR/Cas9-mediated knockout of the eye pigmentation gene white leads to alterations in colour of head spots in the oriental fruit fly, Bactrocera dorsalis.

The intensely studied white gene is widely used as a genetic marker in Drosophila melanogaster. Here, we cloned and characterized the white gene in an important pest of the fruit industry, Bactrocera dorsalis, to understand its functional role in pigmentation. We obtained BdWhite knockout strains, based on the wild-type strain, using the CRISPR/Cas9 genome editing system, and found that mutants lost pigmentation in the compound eye and their black head spots. We then examined differences in the expression levels of genes associated with melanin pigmentation between mutants and the wild-type strain using quantitative reverse transcription PCR. We found that transcription levels of the Bd-yellow1 were lower in the head of mutants than in the wild-type strain, and there were no significant differences in expression of the other six genes between mutants and the wild type. Since yellow is critical for melanin biosynthesis (Heinze et al., Scientific Reports. 2017;7:4582), the lower levels of expression of Bd-yellow1 in mutants led to reduced dark pigmentation in head spots. Our results provide the first evidence, to our knowledge, that white may play a functional role in cuticle pigmentation by affecting the expression of yellow.

Short-term outcomes of laparoscopic ventral rectopexy for obstructed defecation in patients with overt pelvic structural abnormalities-a Chinese pilot study.

AIM: As laparoscopic ventral rectopexy (LVR) gained increasing popularity in the past decade, studies from non-western area remain rare. The aim of this pilot study is to evaluate the efficacy and safety of LVR for obstructed defecation (OD) in Chinese patients with overt pelvic structural abnormalities. METHODS: A series of 19 consecutive patients is presented undergoing LVR for OD. All patients showed various forms of pelvic structural abnormalities which were verified by dynamic defecography exam. Data was prospectively recorded and the functional outcomes were evaluated using the Cleveland Clinic Constipation Score (CCCS) and Patients Assessment of Constipation Quality of Life Score (PAC-QoL). RESULTS: Pelvic structural abnormalities of the 19 patients included external rectal prolapse (ER) in 10.5% (2/19), high grade internal rectal prolapse (IR) in 57.8% (11/19), rectocele in 52.6% (10/19), enterocele in 15.7% (3/19), cystocele/vaginal prolapse in 15.7 (3/19), descending perineum (DP) in 63.5% (12/19). 89.4% patients (17/19) showed at least two co-existed abnormalities and 15.7% (3/19) showed multicompartmental abnormalities. The mean follow-up was 9 months (range 1-18). No mesh-related complication was observed. At last available follow-up (LAFU), improvement of OD symptom was observed in 15 (78.9%) patients, the mean scores of CCCS decreased from 17 to 10 (p < 0.05), all four subsets of PAC-QoL scores improved, and three of them (physical discomfort, worries and concerns, satisfaction) showed statistical significance (p < 0.05). CONCLUSION: Laparoscopic ventral rectopexy appears to be a safe and effective procedure for obstructed defecation in Chinese patients with overt pelvic structural abnormalities in short-term follow-up.

A new family of insect muscarinic acetylcholine receptors.

Most currently used insecticides are neurotoxic chemicals that target a limited number of sites and insect cholinergic neurotransmission is the major target. A potential target for insecticide development is the muscarinic acetylcholine receptor (mAChR), which is a metabotropic G-protein-coupled receptor. Insects have A- and B-type mAChRs and the five mammalian mAChRs are close to the A-type. We isolated a cDNA (CG12796) from the fruit fly, Drosophila melanogaster. After heterologous expression in Chinese hamster ovary K1 cells, CG12796 could be activated by acetylcholine [EC50 (half maximal effective concentration), 73 nM] and the mAChR agonist oxotremorine M (EC50 , 48.2 nM) to increase intracellular Ca(2+) levels. Thus, the new mAChR is coupled to Gq/11 but not Gs and Gi/o . The classical mAChR antagonists atropine and scopolamine N-butylbromide at 100 µM completely blocked the acetylcholine-induced responses. The orthologues of CG12796 can also be found in the genomes of other insects, but not in the genomes of the honeybee or parasitoid wasps. Knockdown of CG12796 in the central nervous system had no effect on male courtship behaviours. We suggest that CG12796 represents the first recognized member of a novel mAChR class.

Determining the age of adult flesh flies, Boettcherisca peregrina, using pteridine fluorescence.

To assess the potential application of pteridine fluorescence in determining the age of adult Boettcherisca peregrina (Diptera: Sarcophagidae) Robineau-Desvoidy and further for the postmortem interval, the age-dependent changes of pteridine fluorescence were investigated for the adults maintained at five constant temperatures. From the results, significant linear relationships were found between pteridine fluorescence and the age of the adults maintained at 16, 20, 24, 28 or 32 °C (P < 0.001, r(2) > 0.85). In addition, the relationships between the rate of pteridine accumulation and temperature were well described using linear equations for adult females and males. Then for each cohort of the flies at the ambient temperature, a calendar was constructed and used to determine the ages of females and males, respectively, in which was recorded in reverse time order the amount of pteridine accumulated per hour by the flies and their expected pteridine level when they emerged at the specified time. A significant linear relationship between estimated ages and chronological ages was observed for female or male adults, with the mean errors of the estimated ages of ±1.82 days for females and ±1.58 days for males. It is suggested that pteridine fluorescence analysis has a potential value in determining the age of adult B. peregrina.

1:4 matched case-control study on influential factor of early onset neonatal sepsis.

BACKGROUND: Bacteria, funghi, viruses and protozoa can lead to neonatal sepsis. Neonatal sepsis is the leading cause of infectious disease onset and death in many neonates. AIM: To explore the major risk factors of early-onset neonatal sepsis and provide a scientific basis for strategies of early-onset neonatal sepsis prevention. SUBJECTS AND METHODS: A 1:4 matched case-control study was adopted and 147 cases of early-onset neonatal sepsis were enrolled. Conditional logistic regression model was used to analyze the univariate and multivariate data to estimate the odds ratio (OR) and the 95% confidence interval (95% CI). RESULTS: Univariate analysis shows that the impact factors on the occurrence of early-onset neonatal sepsis include the following: Maternal age > 35, mother having fixed occupation, mother of urban residence, abnormal fetal position, fetal times, parity, caesarean section, premature rupture of membranes, amniotic fluid volume abnormalities, pregnancy-induced hypertension, placental abnormalities, fetal distress, newborn gender, low birth weight infants, neonatal Apgar scoring at one and five minutes, neonatal jaundice, wet lung, anemia, IVH, and premature infant. Multivariate logistic regression analysis showed that maternal age > 35 (OR = 4.835, OR 95% CI = 1.170-19.981), cesarean section (OR = 0.103, OR 95% CI = 0.041-0.258), premature rupture of membranes (OR = 0.207, OR 95% CI = 0.078-0.547), premature infants (OR = 0.059, OR 95% CI = 0.010-0.329) and newborn jaundice (OR = 0.092, OR 95% CI = 0.021-0.404) were the factors of early-onset neonatal sepsis. CONCLUSIONS: Early-onset neonatal sepsis could be affected by multi-factors, and targeted prevention may reduce the incidence of early-onset neonatal sepsis rates.

Efficacy evaluation of two transgenic maize events expressing fused proteins to CrylAb-susceptible and -resistant Ostrinia furnacalis (Lepidoptera: Crambidae).

The Asian corn borer, Ostrinia furnacalis (GuenCe), is a major pest of maize in China. Transgenic Bt maize could provide an effective way to control this pest. However, the potential of resistance evolution has been documented in the laboratory-a CrylAb-resistant strain of Asian corn borer (Asian corn borer-AbR) could survive well on two Bt maize events, that is, MON810 and Bt11. Pyramided transgenic crops with multiple dissimilar Bt insecticidal proteins offer a superior route for pests control compared with that with one Bt single gene. In this study, two novel transgenic Bt maize events (N30 and V3), which contained a fused gene Cry1Ab/Cry2Aj and CrylAb /Vip3DA, respectively, were evaluated for their efficacy on protection against CrylAb-susceptible Asian corn borer (Asian corn borer-BtS) as well as their potential for dealing with the Asian corn borer-AbR. In laboratory bioassay, no neonate larvae of Asian corn borer-BtS fed on the whorl leaves, tassels, husks, silks, and kernels of N30 and V3 could survive at 96 h after infestation. In contrast, only 2.0-8.7% larvae died as they fed on non-Bt maize tissues. As Asian corn borer-AbR neonate larvae fed on N30 and V3 silks and kernels, 95.0% even more larvae were killed at 96 h after infestation, and all died at 168 h after infestation, while 10.0-11.67% larvae survived on non-Bt control. In the field test under artificial infestation at middle whorl leaf and silking stages of maize, there were no significant leaf, silk, and stalk damage on Bt maize plants by comparison with severe leaf feeding, stalk born, and ear damage in control, in addition, the expression profiles of a fused protein of CrylAb/Cry2Aj or CrylAb/Vip3DA in different tissues of N30 and V3 were also confirmed. In conclusion, it is clear that both Bt maize events (N30 and V3) show great potential for protecting maize from attack by Asian corn borer that has resistance to Cry1Ab protein.

Molecular cloning and developmental expression of the vitellogenin gene in the endoparasitoid, Pteromalus puparum.

A cDNA of the vitellogenin (Vg) protein gene was isolated from the endoparasitoid Pteromalus puparum and characterized. The putative coding sequence was found to be 5634 bp long, encoding 1803 amino acids in a single open reading frame. The chemically determined N-terminal amino acid sequence of vitellin completely matched the deduced amino acid sequence that follows a putative signal peptide of 17 amino acid residues. The Vg mRNA was detected in the fat body of late female pupae, whereas the ovary and male fat body lacked the Vg transcript. The Vg mRNA level in the fat body depended significantly on the developmental stage, reaching the highest level 0 h after eclosion. The haemolymph Vg titre appeared 24 h after the appearance of Vg transcript. A positive correlation between the titre and transcript level of Vg in individual female wasps was found.

The susceptibility testing of 13 strains of Mycobacterium leprae to rifampicin and the determination of minimal effective dosage.

By use of the mouse footpad technique, the susceptibility testing of 13 strains of Mycobacterium leprae to rifampicin (RFP) and the determination of minimal effective dosage (MED) were carried out. Among these strains of M. leprae, 8 were obtained from previously untreated multibacillary leprosy patients and 5 from relapsed leprosy patients without using RFP previously. The results showed that the MED of all strains to RFP were less than or equal to 0.001% FRP in the diet, 5 strains being equal to 0.001%, 5 less than or equal to 0.0001%, 2 greater than or equal to 0.0003% and 1 less than or equal to 0.0003%. The results indicated that the MED value of RFP could be lower than that of other reports. Because the critical concentration of RFP for assessment of RFP-resistant strains is not well established a further study would be worthwhile. The results of the determination of sera RFP concentrations in mice administered the RFP diet were identical with that of Holmes' report. Five of the 13 strains also showed that the growth of bacilli were suppressed by 10 mg/kg RFP using the gavage method.

Studies on mode of detection of leprosy in China during the years 1981-1998.

Along with the nationwide economic reform initiated in the early 1980s and the rapid decrease of leprosy endemic after the implementation of multi-drug therapy (MDT), the leprosy programme changed from 'vertical' to 'horizontal'. An evolution in the mode of detection of leprosy cases has consequently taken place. Based on the nationwide registration of newly detected cases, the profile of patients at detection has been studied. The proportions of cases corrected significantly with calendar years in detection by dermatological clinics, contact checks, 'clue survey' and mass survey, showing a significant increase in percentage of cases detected through dermatological clinics and contact checks, and decreases through 'clue survey' and mass survey. Detection of cases through dermatological clinics and voluntary reporting have become the main modes of case-finding during 1997-1998, accounting for 37.3% and 28.6%, respectively, where contact check accounts for only 9.1%. In areas with good dermatological services, a significantly higher proportion (75.9%) of cases was detected through dermatological clinics, where voluntary reporting and 'clue survey' were the main modes of detection in endemic areas. As regards confirmation of diagnosis, the great majority of cases were confirmed by leprosy units, even though they were detected in various other situations. Only 6.5% of leprosy cases were detected and subsequently confirmed by doctors in dermatologic clinics. The present modes of detection and their relation to demographical, epidemiological, clinical factors and health services are discussed. This study emphasizes the cardinal importance of the dermatological clinics in the detection of leprosy cases in China at the present time and hence the need to strengthen the training of doctors in these clinics, while continuously encourage their involvement in leprosy control.

An investigation of attitudes, beliefs and behaviour of leprosy patients, family members and PHC workers towards multidrug therapy in Yangzhou and Dongtai Districts of China.

To improve the operational efficiency of multidrug therapy (MDT) implementation in rural areas, an investigation into the attitudes, beliefs and behaviour of leprosy patients and their family members as well as primary health care (PHC) workers towards MDT was carried out in Yangzhou and Dongtai Districts of China. A sample of 370 leprosy patients, 594 family members and 730 PHC workers was interviewed or investigated individually using questionnaires. The results showed that: 1, the presently used MDT is acceptable to a wide range of patients although a small number of patients have various problems in their treatment; 2, the patients' habit in daily drug administration, their awareness of the risk of default and confidence in MDT have a positive influence in increasing drug compliance; and 3, the supervision and encouragement of family members to patients' treatment which is associated with their knowledge on MDT is also beneficial to patients' drug compliance. However, only half of the PHC workers had a basic knowledge of MDT and a desire to participate in MDT implementation, a finding which clearly calls for urgent attention and improvement. In order to ensure the effective implementation of MDT, there is a need to educate leprosy patients and their family members as well as PHC workers to establish the patients' correct awareness of MDT, obtain the family support and motivate the PHC workers.

Field evaluation of resistance of transgenic rice containing a synthetic cry1Ab gene from Bacillus thuringiensis Berliner to two stem borers.

Two transgenic rice (Oryza sativa L.) lines, KMD1 and KMD2 at the R4 generation, transformed with a synthetic cry1Ab gene from Bacillus thuringiensis Berliner, were first evaluated for stem borer resistance in the field during the rice growing season of 1998 in two areas of Zhejiang Province, China. Both KMD1 and KMD2 were highly resistant to the stem borers Chilo suppressalis (Walker) and Scirpophaga incertulas (Walker), and were completely undamaged during the whole rice growing season. In contrast, damage to the plants of the untransformed parental control (Xiushui 11) was in the form of deadhearts or whiteheads. Under natural infestation by the C. suppressalis, the damage to control plants reached a peak of 88.7% of plants and 20.1% of tillers encountered with deadhearts. Under artificial and natural infestation of neonate striped stem borers at the vegetative stage and booting stage, 100% of plants and 25.6% of tillers, 78.9% of plants and 15.6% of productive tillers among artificially infested control plants were observed with the symptom of deadhearts and whiteheads, respectively. Damage to the control plants from artificial infestation by the S. incertulas reached a peak of 97.0% of plants and 22.9% of tillers damaged. The field research indicated that both KMD1 and KMD2 show great potential for protecting rice from attack by these two stem borers.

Prediction of elimination of leprosy in leprosy endemic areas of China.

A study was carried out based upon the data from the National System for Leprosy Surveillance and using appropriate mathematical models. The results showed that of 337 counties where the national goal of basic eradication of leprosy had not been reached and in 40 counties where the WHO goal of leprosy elimination had not been achieved in 1996, the detection rates in calendar years followed exponential models with significant goodness-of-fit. In the 67 counties with downward trends of detection rates, the national goal can be met in terms of detection rate in 6% of counties before the year 2000 or 34.4% before the year 2010, or, in terms of prevalence rate in 31.3% before the year 2010. In the 11 counties with downward trends of the detection rates, the WHO target can be met in eight to ten counties within this century when the duration of disease was determined with the WHO definition. If the MB proportion among new cases increased by 10%, the target would be met one year later. However, at the same MB proportion, the change of fixed treatment schedules from PB six months and MB two years to PB nine months and MB three years will cause achievement of the goal to be postponed by two to ten years.

Impacts of Bt rice expressing Cry1C or Cry2A protein on the performance of nontarget leafhopper, Nephotettix cincticeps (Hemiptera: Cicadellidae), under laboratory and field conditions.

Transgenic rice expressing Bacillus thuringiensis Berliner (Bt) protein can effectively control target insects including stem borers and leaf folders. However, the potential effects of Bt rice on nontarget organisms including nontarget herbivores have not been fully evaluated. In the current study, ecological fitness parameters of the nontarget herbivore, Nephotettix cincticeps (Uhler) (Hemiptera: Cicadellidae), fed on T1C-19 (Cry1C) or T2A-1 (Cry2A) rice were compared with non-Bt rice (MH63) under laboratory conditions. A 2-yr field trial was also conducted to monitor the population dynamics of N. cincticeps in the Bt and control rice plots using the vacuum-suction machine and yellow sticky card traps. Laboratory results showed that there were no significant differences in some of biological parameters including egg developmental duration, adult fresh weight, adult longevity, and oviposition period when N. cincticeps fed on Bt or non-Bt rice was compared. However, the survival rate of N. cincticeps nymphs fed on T2A-1 Bt rice plants was significantly higher than that on the control. When N. cincticeps fed on T1C-19 Bt rice plants, its nymphal duration was significantly longer and fecundity significantly lower compared with those fed on both T2A-1 Bt and non-Bt rice plants; the preoviposition period of N. cincticeps fed on T1C-19 and T2A-1 Bt rice was also significantly shorter than those on non-Bt rice. Nonetheless, both seasonal density and population dynamics of N. cincticeps adults and nymphs were similar between Bt (T1C-19 and T2A-1) and non-Bt rice plots under field conditions. In conclusion, our results indicate that our two tested Bt rice lines would not lead to higher population of N. cincticeps. Long-term experiments to monitor the population dynamics of N. cincticeps at large scale need to be carried out to confirm the current results.

No direct effects of two transgenic Bt rice lines, T1C-19 and T2A-1, on the arthropod communities.

A 2-yr field trial was conducted to assess the impacts of two new transgenic Bt rice lines, T1C-19 expressing Cry1C protein and T2A-1 expressing Cry2A protein, on the arthropod community sampled via vacuum. All the arthropods were classified into five guilds, including herbivores, parasitoids, predators, detritivores, and others. The seasonal density and dominance distribution of each guild and community-level indices (species richness, Shannon-Wiener diversity index, Simpson diversity index, and evenness index) were compared among rice types. Principal response curves were used to investigate the differences of entire arthropod community of Bt rice plots relative to non-Bt rice plots. The results showed no significant difference was detected in the community-level indices and dominance distribution of guilds between Bt and non-Bt rice plots. The seasonal density of herbivores, detritivores, and others as well as density of the arthropod overall community were also not significantly affected by rice types in either year, although the density of predators and parasitoids in Bt rice plots was significantly lower than those in non-Bt rice plots. The lower abundances of Braconidae, Eulophidae, Cyrtorhinus lividipennis (Reuter) (Hemiptera: Miridae), and Theridiidae in Bt rice plots are likely attributed to the lower abundances of prey species or hosts. Principal response curves revealed that arthropod community in Bt was similar with that in non-Bt rice plots. In conclusion, our findings indicate that these two tested Bt rice lines had no marked negative effects on the arthropod community in the paddy fields.

Impact of six transgenic Bacillus thuringiensis rice lines on four nontarget thrips species attacking rice panicles in the paddy field.

As a key component of ecological risk assessments, nontarget effects of Bacillus thuringiensis (Bt) rice have been tested under laboratory and field conditions for various organisms. A 2-yr field experiment was conducted to observe the nontarget effects of six transgenic rice lines (expressing the Cry1Ab or fused protein of Cry1Ab and Cry1Ac) on four nontarget thrips species including Frankliniella intonsa (Trybom), F. tenuicornis (Uzel), Haplothrips aculeatus (F.), and H. tritici (Kurd), as compared with their rice parental control lines. Two sampling methods including the beat plate and plastic bag method were used to monitor the population densities of the four thrips species for 2 yr. The results showed that the seasonal average densities of four tested thrips species in Bt rice plots were significantly lower than or very similar to those in the non-Bt rice plots depending on rice genotypes, sampling methods, and years. Among all six tested Bt rice lines, transgenic B1 and KMD2 lines suppressed the population of these tested thrips species the most. Our results indicate that the tested Bt rice lines are unlikely to result in high population pressure of thrips species in comparison with non-Bt rice. In some cases, Bt rice lines could significantly suppress thrips populations in the rice ecosystem. In addition, compatibility of Bt rice, with rice host plant resistance to nontarget sucking pests is also discussed within an overall integrated pest management program for rice.

Impacts of six bt rice lines on nontarget rice feeding thrips under laboratory and field conditions.

Nontarget impacts of six transgenic Bt rice lines (expressing the Cry1Ab or Cry1Ab/Cry1Ac protein) on the thrips, Stenchaetothrips biformis (Bagnall), attacking the rice seedling and tillering stages, were evaluated under laboratory and field conditions. Laboratory results showed relatively longer larval, pupal development and preoviposition durations of S. biformis. Although it had a shorter oviposition period, female adult longevity and less total laid eggs were found when fed on some tested Bt rice in comparison to non-Bt controls. S. biformis population dynamics in Bt and non-Bt plots were monitored using the plastic bag and beat plate methods. In the field, the temporal patterns of S. biformis population changes were similar between tested Bt rice lines and their respective control; however, the total number of S. biformis individuals collected from the Bt plots were significantly less or the same, varying from variety to variety, compared with those from the non-Bt plots. ELISA results showed that the Bt insecticidal protein could be transferred from Bt rice to the thrips, and the concentrations of the protein in rice leaves and thrips were not significantly correlated with some important biological parameters of the thrip. In addition, the potential effects of Bt rice on the abundance of S. biformis candidate predators are also discussed. In conclusion, our results show that the six Bt rice lines assessed may be less preferable host plants to S. biformis at the individual and population levels in comparison to the non-Bt rice plants.

Effects of transgenic rice expressing Bacillus thuringiensis Cry1Ab protein on ground-dwelling collembolan community in postharvest seasons.

During 2005-2008, field studies were conducted at two locations in Chongqing, China, to assess the potential effects of transgenic rice expressing Bacillus thuringiensis (Bt) Cry1Ab protein on the nontarget ground-dwelling collembolan community in three postharvest seasons. Collembolans in non-Bt and Bt rice fields were sampled with pitfall traps during each of two postharvest seasons of 2005/2006 and 2006/2007 and litterbag traps during each of three postharvest seasons of 2005/2006, 2006/2007, and 2007/2008. Ground-dwelling collembolans in rice fields during the postharvest seasons were abundant, whereas community densities varied considerably between the two locations and among the three seasons. A total of 67,310 collembolans, representing three species, Entomobrya griseoolivata, Hypogastrura matura, and Bourletiella christianseni, were captured during the three postharvest seasons. E. griseoolivata was the predominant species, accounting for 87.7% of the total captures, followed by H. matura (10.7%) and B. christianseni (1.6%). In general, there were no significant differences in species compositions and abundances of each species between Bt and non-Bt paddy fields, suggesting no significantly impact of plant residues of Cry1Ab rice on collembolan communities during postharvest seasons.

Laboratory and field assessments of prey-mediated effects of transgenic Bt rice on Ummeliata insecticeps (Araneida: Linyphiidae).

One major concern regarding the release of Bt rice is its potential impact through tritrophic interactions on nontarget arthropods, especially natural enemies. We studied the effects of two Bt transgenic rice varieties, TT9- 3 and KMD1, expressing Cry1Ab/Cry1Ac and Cry1Ab, respectively, on a predatory ground spider [Ummeliata insecticeps (Bösenberg et Strand)] supplied with Bt rice-fed brown planthopper [Nilaparvata lugens (Stål)] nymphs. Although immunoassays confirmed that U. insecticeps ingested Bt insecticidal protein when supplied with Bt rice-fed N. lugens, no negative effects were found on its survival and development. Furthermore, the fecundity of U. insecticeps fed prey reared on Bt rice was not significantly different from that of those fed prey reared on non-Bt rice. A 3-yr field trial indicated that Bt rice did not significantly affect the population density of U. insecticeps in comparison with non-Bt rice. In conclusion, the Bt rice lines tested in this study had no adverse effects on the survival, developmental time, or fecundity of U. insecticeps in the laboratory or on population dynamics in the field.