Backcrosses of XX and XY males from dsRNA injected eggs were backcrossed to wild females in 35 cages.

<p>^aThe percentage of Female-only emerging is calculated by dividing into the number of cages with adults emerging.</p><p>Backcrosses of XX and XY males from dsRNA injected eggs were backcrossed to wild females in 35 cages.</p

Phenotypes analyses of RNAi intersex individuals.

<p>(A) Wild-type male with 25 black short bristles on each lower side of the third tergum, magnification of the red dotted section in A is shown in A’; (B) Intersex individual with male genitalia and female dorsal phenotype without bristles on both sides; (C) Normal male genitalia in XY males; (D) Malformed male genitalia of one intersex individual; (E) One XY male with an excessive row of bristles on the right lower side of the second tergum, magnification of the red dotted section in E was shown in E’. A, B, E: dorsal view; C-D: ventral view; arrows indicate the position of bristles.</p

Expression patterns of <i>Bdtra</i> and <i>Bdtra-2</i>.

<p>(A, B) RT-PCR with <i>Bdtra</i> primers 225+/706- yielded male and female products with different sizes as indicated. RT-PCR with <i>Bdtra-2</i> primers 9+/1185- yielded male and female products of the same size. (C) RT-PCR with male-specific primer ms43+ and non-sex specific primer 706- to amplify the male-specific product. M, 100 bp DNA ladder (Takara); E0–E8, mixed sex embryos collected 0–0.5 h, 0.5–1 h, 1–2 h, 2–4 h, 4–6 h, 6–8 h, 8–10 h, 10–14 h, 14–21h and 24–48 h after egg laying, respectively; L1, first instar larvae of mixed sex; L3, third instar larvae of mixed sex; P, newly pupated pupae of mixed sex; 0d♀,newly emerged female adult; 0d♂, new emerged male adult; ♀md, midguts from 0d♀; ♂md, midguts from 0d♂; ♀fb, fat bodies from 0d♀; ♂fb, fat bodies from 0d♂; ov, ovaries; te, testes; <i>a-tub</i>, reference gene <i>a-tubulin</i>.</p

RNAi-Mediated Knock-Down of <i>transformer</i> and <i>transformer 2</i> to Generate Male-Only Progeny in the Oriental Fruit Fly, <i>Bactrocera dorsalis</i> (Hendel)

<div><p>The <i>transformer</i> (<i>tra</i>) gene appears to act as the genetic switch that promotes female development by interaction with the <i>transformer2</i> (<i>tra-2</i>) gene in several dipteran species including the Medfly, housefly and <i>Drosophila melanogaster</i>. In this study, we describe the isolation, expression and function of <i>tra</i> and <i>tra-2</i> in the economically important agricultural pest, the oriental fruit fly, <i>Bactrocera dorsalis</i> (Hendel). <i>Bdtra</i> and <i>Bdtra-2</i> are similar to their homologs from other tephritid species. <i>Bdtra</i> demonstrated sex-specific transcripts: one transcript in females and two transcripts in males. In contrast, <i>Bdtra-2</i> only had one transcript that was common to males and females, which was transcribed continuously in different adult tissues and developmental stages. <i>Bdtra-2</i> and the female form of <i>Bdtra</i> were maternally inherited in eggs, whereas the male form of <i>Bdtra</i> was not detectable until embryos of 1 and 2 h after egg laying. Function analyses of <i>Bdtra</i> and <i>Bdtra-2</i> indicated that both were indispensable for female development, as nearly 100% males were obtained with embryonic RNAi against either <i>Bdtra</i> or <i>Bdtra-2</i>. The fertility of these RNAi-generated males was subsequently tested. More than 80% of RNAi-generated males could mate and the mated females could lay eggs, but only 40-48.6% males gave rise to progeny. In XX-reversed males and intersex individuals, no clear female gonadal morphology was observed after dissection. These results shed light on the development of a genetic sexing system with male-only release for this agricultural pest.</p></div

Statistics on knock-down phenotypes of <i>Bdtra</i> and <i>Bdtra-2</i> by dsRNA injection.

<p>^aThe percentage of the phenotypes is divided into the number of adults obtained from each treatment.</p><p>Statistics on knock-down phenotypes of <i>Bdtra</i> and <i>Bdtra-2</i> by dsRNA injection.</p

Internal genital structures of RNAi males.

<p>Testes of XY males (A) and testes that demonstrated various types of abnormal morphology such as a well-developed testis plus a poorly developed testis (B), hypertrophic testes (C, F), underdeveloped testes (D), single testis (E) from XX-reversed males and intersex individuals. Similar results were obtained for ds<i>Bdtra</i> and ds<i>Bdtra-2</i> injections. Scale bars are all the same and indicate 0.2mm.</p

Expression patterns of <i>Bdtra</i> and <i>Bddsx</i> in RNAi males.

<p>(A, B) Injection with ds<i>Bdtra</i>_79. (C, D) Injection with ds<i>Bdtra</i>_613. (E, F) Injection with ds<i>Bdtra-2</i>_422. (A, C, E) Detection of the transcripts of <i>Bdtra</i>. (B, D, F) Detection of the transcripts of <i>Bddsx</i>. Abbreviations and notes: <i>Bdtra</i> M and <i>Bddsx</i> M: male-specific splicing forms of <i>Bdtra</i> and <i>Bddsx</i> respectively; <i>Bdtra</i> F and <i>Bddsx</i> F: female-specific splicing forms of <i>Bdtra</i> and <i>Bddsx</i> respectively; <i>a-tub</i>, reference gene a-<i>tubulin</i>. The band between <i>Bddsx</i> M and <i>Bddsx</i> F was a male transcript with the size of 130bp shorter than <i>Bddsx</i> M in 3’UTR. Lane 1: female fly from ds<i>EGFP</i>-injection; Lane2: male fly from ds<i>EGFP</i>-injection; Lane 3, 4, 5: XX-reversed males; Lane 6, 7, 8; intersex individuals; Lane M: Takara 100bp DNA ladder.</p

Genomic and cDNA structure of <i>Bdtra</i> and <i>Bdtra-2</i>.

<p>(A) Genomic structure of <i>Bdtra</i> showing exons (boxes), introns (lines), sex-specific transcripts in males and females and the locations of the Tra/Tra-2, RBP1 and ISS binding sites. (B) Genomic structure of <i>Bdtra-2</i> showing exons (boxes), introns (lines) and the non-sex-specific transcript. (C) The consensus sequences of Tra/Tra-2, RBP1 and ISS binding sites. Horizontal arrows indicate the positions of primer pair 225+/706- and ms43+/706- (A) and primer pair 9+/1185- (B) for the expression analyses and numbers down the lines and inside the boxes represent the length of introns and exons, respectively. Vertical bars represent the position of translation start codons. Red boxes stand for the male-specific exons with the translation stop codons marked by asterisks above. Blue horizontal lines indicate the positions of the double strand RNAs named ds<i>Bdtra</i>_79, ds<i>Bdtra</i>_613 (A) and ds<i>Bdtra-2</i>_422 (B) for RNAi.</p

Table1_Apomixis for no bacteria-induced thelytoky in Diglyphus wani (Hymenoptera: Eulophidae).xlsx

In Hymenoptera species, the reproductive mode is usually arrhenotoky, where haploid males arise from unfertilized eggs and diploid females from fertilized eggs. In addition, a few species reproduce by thelytoky, where diploid females arise from unfertilized eggs. Diploid females can be derived through various cytological mechanisms in thelytokous Hymenoptera species. Hitherto, these mechanisms were revealed mainly in endosymbiont-induced thelytokous Hymenoptera species. In contrast, thelytokous Hymenoptera species in which a reproductive manipulator has not been verified or several common endosymbionts have been excluded were paid less attention in their cytological mechanisms, for instance, Diglyphus wani (Hymenoptera: Eulophidae). Here, we investigated the cytological mechanism of D. wani using cytological methods and genetic markers. Our observations indicated that the diploid karyotypes of two strains of D. wani consist of four pairs of relatively large metacentric chromosomes and one pair of short submetacentric chromosomes (2n = 10). The arrhenotokous strains could complete normal meiosis, whereas the thelytokous strain lacked meiosis and did not expulse any polar bodies. This reproductive type of lacking meiosis is classified as apomictic thelytoky. Moreover, a total of 636 microsatellite sequences were obtained from thelytokous D. wani, dominated by dinucleotide repeats. Genetic markers results showed all three generations of offspring from thelytokous strain maintained the same genotype as their parents. Our results revealed that D. wani is the first eulophid parasitoid wasp in Hymenoptera whose thelytoky was not induced by bacteria to form an apomictic thelytoky. These findings provide a baseline for future inner molecular genetic studies of ameiotic thelytoky.</p

Levels of Salivary Enzymes of <i>Apolygus Lucorum</i> (Hemiptera: Miridae), From 1^st Instar Nymph to Adult, and Their Potential Relation to Bug Feeding

<div><p>In recent years, <i>Apolygus lucorum</i> has caused increasing damage to cotton and fruit trees in China. The salivary enzymes secreted by <i>A</i>. <i>lucorum</i> when sucking on host plants induce a series of biochemical reactions in plants, and the pre-oral digestion benefits the bug feeding. In this study, the food intake of <i>A</i>. <i>lucorum</i> from 1^st instar nymphs to adults was measured, and the corresponding salivary activity of pectinase, amylase, cellulase, protease, polyphenol oxidase and peroxidase was determined. Daily food intake varied with developmental stage, peaking in 3^rd and 4^th instar nymphs. Pectinase, amylase, cellulase and protease were detected in both nymphal and adult saliva of <i>A</i>. <i>lucorum</i>, while neither polyphenol oxidase nor peroxidase was detected. Protease activity varied with food intake peaking at the 3^rd-4^th instar, and then slightly decreasing at the 5^th instar. Levels of pectinase, amylase and cellulase increased significantly with the daily feeding level until the 3^rd instar, corresponding with increasing damage to host plants. The activity of both cellulase and protease had a significant linear relationship with the average daily food intake. The increasing activity of enzymes in saliva explain stage-specific impacts of <i>A</i>. <i>lucorum</i> on the host plants, and suggest that optimal management of <i>A</i>. <i>lucorum</i> would be confined to its control threshold prior to the peak of daily feeding in the 3^rd instar.</p></div