sexual development in insect vectors

Question 1

sex of arthropods

Answer 1

• sexual dimorphism
  
  • drastic differences in body depending on which chromosomes are present
  • imapacts disease tranmission
  • usually female transmission
• identify genes involved to manipulate sterility
  
  • distort sex ratio by SIT etc.

Question 2

sex chromosomes of insects

Answer 2

• very few genes
• males are heterogametic
  
  • mammals = SRY gene that determines maleness
• presence of Y in drosophila doesn’t determine sex

Question 3

sex determination in drosophila

Answer 3

• X to autosome ratio determines sex
• 2 sets of autosomes, 2 sex chromosomes
• female: X:A = 1.0 (or greater)
  
  • XX and autosomes
• male: X:A =0.5 (or less)
  
  • XY and autosomes
• intersex: X:A between 0.5 and 1.0

Question 4

X and Y chromosome of drosophila

Answer 4

• X contains genes determining femaleness
  
  • requires the correct ratio to autosomes
  • single X chromosome (no Y) produces males
• presence of Y alone does not determine maleness
  
  • XXY doesn’t produce males

Question 5

dosage compensation in drosophila

Answer 5

• occurs in mammals to equilibriate levels of genes on X between males and females
  
  • X-inactivation - 1 X silenced
• not conserved in drosophila
  
  • X hypertranscription in males to compensate
• in nematodes, X hypotranscription in females occurs

Question 6

downstream of X:A ratio detection

Answer 6

• activation of sex-specific splicing cascade
  
  • different sex splicing factors produced
  • in drosophila:
    
    • sex lethal
    • transformer
    • doublesex

Question 7

sex lethal

Answer 7

• sxl gene with 3 exons 2 introns
  
  • male → non-functional product
  • female → functional sxl due to presence of female-specific splicing factor
• sxl splicing factor regulates transformer

Question 8

transformer

Answer 8

• tra gene
• differentially spliced in males and females due to presence/absence of functional sxl
• males → nonfunctional
• females → functional transformer splicing factor produced
  
  • acts on doublesex

Question 9

doublesex

Answer 9

• dsx gene for TF
• different C-terminus in males and females due to alternative splicing from tra presence/absence
• male product → represses female development genes
• female product → represses male development genes
• mainly repsonsible for morphological differences
  
  • somatic sexual differentiation
    
    • genitalia

Question 10

conservation of sxl and tra

Answer 10

• not conserved across insects
• ceratitis capitata
  
  • sxl gene present but not involved in pathway
• rapidly evolving
  
  • difficult to identify by homology
• complexity of alternative splicing is conserved
  
  • always involved in leading to functional cascade elements

Question 11

fruitless

Answer 11

• splicing dependent on tra
• functional tra →nonfunctional fruitless → female nervous system development
• nonfunctional tra → functional fruitless → male nervous system development and behaviour
  
  • includes mating behaviour

Question 12

key morphological phenotypes in males and females

Answer 12

• males:
  
  • large dark colouration (genitalia)
  • sex comb on one leg to trap females
• females:
  
  • differential badning pattern in terminal segments
  • vulva
  • absence of sex comb

Question 13

mutation of transformer

Answer 13

• genetic background of female with mutated transformer
• different male phenotype
  
  • sex comb, altered badning pattern, male-like genitalia

Question 14

altered dsx expression

Answer 14

• genetic male with female dsx expression
  
  • disappearance of sex comb
  • terminal dark colouration
  • female-like genitalia

Question 15

fruitless misexpression

Answer 15

• site-specific engineering to replace endogenous fruitless with differently spliced forms
  
  • replace with WT locus → same WT phenotype
• replace with reverse transcribed cDNA from RNA of male/female form
  
  • female fruitless → no expression
  • male fruitless → fruitless in both males and females
• knockout upstream tra → fruitless in males and females
  
  • no regulation of splicing

Question 16

male courtship behaviour

Answer 16

• specific steps performed by male
  
  • response to olfactory and visual female clues
• males with nonfunctional fruitless
  
  • chase each other instead of females
  • male biology but female behaviour
• females with functional fruitless
  
  • form chains by trying to mate with other females
  • female biology but male behaviour

Question 17

applications in SIT

Answer 17

• male tra has 2 splicing events
  
  • truncated protein with early stop codon
• female tra has long intron and exons removed
• put alternatively spliced tra gene region in toxic gene
  
  • same splicing pattern occurs in tra
  • female → toxic gene is functional due to splicing out of specific region
  • male → nonfunctional truncated toxic protein
• can produce largely male populations as females die

Question 18

sex development in anopheles

Answer 18

• tra not identified
• dsx and fruitless homologues identified
• XXY is male (unlike drosophila)indication of Y-linked male factor like mammals

Question 19

dsx in anopheles

Answer 19

• 1 female-specific isoform an 1 male isoform
• share first 4 exons
  
  • 5th is unique in each
• use of splicing to create sexing strain

Question 20

anopheles sexing strain

Answer 20

• ubiquitous promoter and GFP gene in spliced exons
• surround with female-specific exons should produce GFP in females only
• saw opposite
  
  • more complex than thought
  • some genome annotations show more than 1 transcript in each sex

Question 21

vasa

Answer 21

• RNA binding protein
  
  • RNA-dependent helicase activity
  • essential for germ cell development
  • promoter used to visualise germ line development with GFP
• homologue in anopheles identified

Question 22

anopheles orthologue of vasa

Answer 22

• used drosophila sequence search
• GFP in upstream regions to visualise ovary/testes development
• at L2 larval stage you can see germ line development
• dimorphism in gonads begins in early larval development

Question 23

studying differential sex-specific transcription

Answer 23

• microarrays
• segregate differentially expressed genes accoridng to time of expression
• rank according to sex-bias size
• cluster
• male bias cluster at late larval stage
  
  • genes involved in sperm formation (meiosis)
  • RNAi knockdown → no testes

Question 24

advantage of NGS over microarrays

Answer 24

• sensitivity
• no need to know genes beforehand to spot
  
  • no bias against non-annotated/spotted genes
• picks up sex-specific splicing alternatives
• also non-coding small RNAs
  
  • microRNAs and siRNAs

Question 25

differences in dsx isoforms

Answer 25

• very similar overall
• C-terminal region differs
• bind same sites but recruit different downstream TFs using terminal domains
• overall conserved structure across many organisms

Question 26

dsx in A. gambiae

Answer 26

• 85 Kb region on 2R
• 7 exons
  
  • exon 5 spliced out in males
• CRISPR/cas9 induced ds breaks in exon 5
  
  • insert GFP → shouldn’t be expressed in males
  • in females → should be disrupted dsx but GFP
  • RFP gene insertion on Y to track Y chromosomes
• cross heterozygotes for disruption

Question 27

disrupted A. gambiae dsx

Answer 27

• heterozygote crosses
  
  • 25% no mutation
  • 50% single copy dsx
  • 25% 2 copies of dsx mutant
• females with disrupted exon 5 (disrupted dsx)
  
  • both copies → bushier antenna, larger intersex palps, claspers for copulation = male somatic development
  • also accessory glands
  • still behave as females
    
    • in tact fruitless