Sex determination refers to the developmental programme that commits the embryo to either the male or the female pathway. The animal kingdom possesses a wealth of mechanisms via which gender is decided, all of which are represented among the insects.

1. SEX DETERMINATION
IN
INSECTS
♀ or ♂ ?
Dr. Devina Seram, Ph.D.
DEPARTMENT OF AGRICULTURAL ENTOMOLOGY

2. What is sexual reproduction?
Process resulting in genetic exchange, variation, and
diversity within a population forming a new embryo
by the mechanisms of segregation and
recombination during meiosis
• Understanding sex determination and sex allocation in
insects has both fundamental and applied applications

3. Why sexual reproduction persists?
1. It provides an ability to incorporate and
accumulate favourable mutations, reducing
mutation load
2. It may allow the accumulation of favourable
mutations and eliminate harmful mutations
(Muller’s ratchet effect)
 Thus, its value “lies more in the ability to
reassort existing genes as the environment
changes and in the elimination of harmful
mutations” (Crow, 1994)

4. Muller’s Ratchet effect
• Muller (1964): In an asexual population, it is
unlikely that any individual is free of harmful
mutations. In such a population, the most fit
individual is one that has only one mutation
• Whereas, a mutant-free type can be created by
recombination in a sexual population

5.  For most diploid eukaryotes, sexual reproduction is
the only mechanism resulting in new members of a
species.
 Meiosis in the sexual organs of parents produces
haploid gametes, which unite during fertilization to
restore the diploid phenotype in the offspring.
 For most organisms, sexual reproduction requires
some form of sexual differentiation.
 In higher forms of life, this is manifested as
phenotypic dimorphism between males and females
of a species.

6. Sex determination in insects
1. Regulates development of a significant character in
eukaryotes (insects)
2. Provide useful tools for the genetic improvement of
arthropod natural enemies of pest arthropods and
weeds and for genetic modification of pests
3. Involves soma and germ-line tissues (ovaries and
testes)
 Sexual dimorphism in adult insects is often extreme,
with differences in setal patterns, pigmentation,
external genitalia, internal reproductive systems, and
behavioral patterns

7. Sex determination in D. melanogaster
• Number of genes involved is relatively low
Involves 3 components:
1) Dosage compensation of X chromosome
2) Somatic sexual development
3) Germ-line sexual development
 Important feature : the number of (X)
chromosomes relative to autosomes (A) in a cell
(X:A ratio)

8. Dosage Compensation
• A basic aspect of sex determination in insects with
an XY sex determining system is dosage
compensation of the X chromosomes
• In other words, it is the mechanism that keeps
females (XX) from expressing twice as much of
X-chromosome genes as males (XY), who have
only one X chromosome
• It equalizes the amount of gene products produced
by individuals with an XX/XY genetic system (males
containing one and females two X chromosomes)
 Its mechanism varies in different arthropods

9. In Drosophila
melanogaster
Achieved by hypertranscription of the
single X chromosome in males
In Gryllotalpa
fossor
Analogous to mammals
- one of the two X chromosomes in
females is transcriptionally inactivated;
males are XO and females are XX
Dosage Compensation
HYPERTRANSCRIPTION - A mechanism of dosage
compensation in the male Drosophila by upregulation of
transcription of the genes in the single X chromosome, thus making
the phenotypes in males (XY) similar to females (XX). It is mediated
by the MSL proteins, which seem to be required for the accumulation
of histone H4, acetylated at lysine 16 in the X of the male

10. Dosage Compensation in D. melanogaster

11. Somatic Sex Determination
• The relative number of X chromosomes and
autosomes is responsible for the primary step in sex
determination immediately after fertilization
• Eg.
• Takes place in individual cells (cell autonomous sex
determination) – due to lack of sex hormones
Cells with two X chromosomes and
two sets of autosomes (2X:2A, or a
ratio of 1.0)
Develop into
females
Diploid cells with one X
chromosomes and two sets of
autosomes (1X:2A, or a ratio of 0.5)
Develop into males
Flies with an intermediate X:A ratio
(XX:AAA)
Develop as
intersexual

12. Somatic sex determination in D. melanogaster

13. • It is regulated through a cascade of sex-specific
events in which RNA transcripts are differentially
processed in males and females
• Sex-lethal+ gene - a key switch gene that affects
both somatic sexual differentiation and dosage
compensation
- regulates pre-mRNA splicing for itself and for
transformer+ (tra+) and male-specific-lethal-2+
(msl-2+)
Somatic sex determination

14. Role of Maternal genes in progeny development
Function in two ways:
1. Mother produces a gene product then transferred
to and stored in the egg
2. Mother’s mRNA is transferred to and stored in the
egg, subsequently translated by the embryo
 Involves four maternal “X:A signal transduction”
genes
a) daughterless+ (da+) b) hermaphrodite+ (her+)
c) extramachrochaetae+ (emc+) d) groucho+ (gro+)

15. Germ Line Sex Determination
• Pole cells in embryo are segregated into the posterior
pole of insect embryo before blastoderm formation
including the progenitors (stem cells) of the germ
cells
• During embryogenesis, male and female germ cells are
indistinguishable - differentiation starts during larval
stage, when male gonads grow larger than female due to
more germ cells
• Sexual identity of germ cells is determined by
both the X:A ratio of the germ cells and the X:A
ratio of the surrounding soma

16. Diverse sex determination mechanisms
in insects
Genetic sex determination system - genetic
differences determining maleness or femaleness
Eg. In D. melanogaster
Environmental sex determination - no genetic
differences between males and females but
temperature or host conditions determine the sex
Eg. In few insects, haemolymph of mother determines
the sex of offspring

17. Ploidy levels in sex determination
1. Both sexes of many
arthropods are diploid
2n, diplo-diploidy
2. Haploid males and diploid
Females
n and 2n, haplo-
diploidy or Arrhenotoky
3. Diploid females 2n or Thelytoky
4. Haploid males produced by loss of
paternally derived chromosomes
after fertilization (some species)
Parahaploidy
5. Males in apterygote and many
pterygote insects are Heterogametic
Males are XO, XY,
XXO, XXY, or XYY
Females are XX
6. Higher pterygotes
(Trichoptera, Lepidoptera)
Females may be
Heterogametic (ZW)

18. Sex Determination Models
Models proposed for haplo-diploid Hymenoptera :
1. Single locus, multiple-allele model – sex determined by
a series of alleles at a single locus
Eg. Apis mellifera, Bracon hebetor
2. Multiple-locus, multiple-allele model – sex determined
by a number of alleles at a series of loci
- females must be heterozygous at one or more loci, while
haploid males are hemizygous

19. Sex Determination Models

20. 3. Genic balance sex determination model – sex
determined by a balance between non-additive male-
determining genes and additive female-determining genes
Eg. Arrhenotokous hymenopterans
INTRASPECIFIC VARIABILITY
 Occurrence of different sex-determining mechanisms within a
single species
Eg.
Sex Determination Models
M. Domestica
(Strain 1)
Sex determined by the presence or absence of
Y, which carries a male-determining factor, M;
X plays no important role in sex determination
M. Domestica
(Strain 2)
Both males and females are XX and have a
special autosome that may carry a male-
determining factor AM that determines sex

21. • Haplo-diploid insects adjust sex ratio of their progeny
based on environmental factors
Eg.
Postzygotic Sex Determination
 Sex determination after the zygote formation; (not at
syngamy)
Eg. In collembolans, Sminthurus viridis and Allacma
fusca, sexes differ with 10 chromosomes in males and
12 in females
Environmental Effects on Sex Determination
Encarsia females
(Hymenoptera:
Aphelinidae)
Develop as autoparasitoids of whiteflies
(primary hosts)
Encarsia males Develop as parasitoids of Encarsia female
pupae (secondary hosts)

22. Single Model for Sex Determination
 Proposed by Nothiger and Steinmann-Zwicky (1985)
 States that - “all the sex determination mechanisms in
insects are variations upon a theme”
• It involves:
a) a gene equivalent to Sxl+,
b) a repressor (R) which inactivates Sxl+,
c) a gene which activates Sxl+,
d) a gene equivalent to dsx+ which is expressed in two
alternative forms

23. Single Model for Sex Determination

24. Sex Determination systems in mosquitoes
In Aedes spp. Sex determined by a dominant male-
determining factor
In Culex spp. Sex determined by a single gene on an
autosome
In Anopheles
gambiae and A.
culicifacies
XY males and XX females present
In Aedes aegypti
and Culex
pipiens
Intersex flies with phenotypes similar to the ix,
dsx, and tra mutants of Drosophila found
In Aedes spp.
(Northern strain)
Sex determination depends upon temperature
at which they are reared, with males
transformed into intersexes at higher
temperatures
Nothiger and Steinmann-Zwicky’s model – Also accounts for sex
determination in haplo-diploid Hymenoptera

25. Distortion of Sex Ratios (♂:♀)
 Occurs when sex chromosome allocation is
disrupted during “meiotic drive”
• Meiotic drive alters the assortment of chromosomes
during meiosis so that certain chromosomes are
inherited more frequently than expected (>50%)
• Eg. In Dipterans - D. melanogaster, Aedes and Culex.
 Includes :
1. Segregation Distorter (SD) gene in Drosophila
2. Male drive in A. aegypti and C. quinquefasciatus
3. Meiotic drive (female drive) in stalk-eyed flies

26. Segregation Distorter (SD) gene in Drosophila
 SD phenotype present at low but stable frequencies
in most field populations
 Occurs when the nuclei of the sperm with the normal
SD+ chromosome fail to condense normally at sperm
maturation
 SD “locus” consists of two overlapping genes:
1) HS2ST and 2) RanGAP
 RanGAP – an essential component for proteins and
RNA molecules transport into and out of the
cell’s nucleus

27. “Distorter” gene in Mosquitoes
(Male drive in A. aegypti and C. quinquefasciatus)
 Distorter gene (D) is linked closely to sex locus
“m/M” and causes chromosome breakage
 Results in production of excess males due to breakage
of X chromosomes during meiosis in males,
 Fewer X than Y chromosomes are transmitted in the sperm,
leading to production of fewer female embryos
 Sensitivity to Distorter controlled by “m”, the female-
determining locus
Eg. Found in mosquito populations from Africa,
America, Australia, Sri Lanka

28.  Occurs due to a meiotic drive element on the X-
chromosome
Eg. In Cyrtodiopsis dalmanni and C. whitei (Diopsidae)
 Eye stalks more exaggerated in males than females
- females prefer to mate with males with a long eye
span
 Long stalks - indication that the male either lacks
meiotic drive or can suppress the meiotic drive
“Distorter” gene in Flies
(Female drive in Stalk-Eyed Flies)

29. Hybrid Sterility (Haldane’s Rule)
 When different species are crossed, hybrid progenies
are produced. However, these progenies may have
altered sex ratios, either one sex absent, rare, or sterile.
 The missing or sterile sex is usually the
heterogametic sex (Laurie, 1997)
 This phenomenon is known as Haldane’s Rule
• Eg. In Drosophila, Musca domestica, Glossina
morsitans, Tetraneura ulmi, etc.

30. “Medea” gene in Tribolium sp.
 A new class of selfish genes – “Medea”
 Causes a “Maternal-Effect Dominant Embryonic
Arrest” - results in the death of zygotes that do not
carry this gene – Tribolium castaneum
• Hypothesis – “Medea” gene led to reproductive
isolation and speciation in T. castaneum
• “Medea” factor absent in T. castaneum populations
of India due to presence of a hybrid incompatibility
factor (H)

31. Sex Ratio Distortion in Arthropods by Cytoplasmic Agents
 “Cytoplasmic sex ratio distorters” – can manipulate
their host’s sex ratio and promote their own spread
 Altered sex ratios in Diptera, Heteroptera, Coleoptera,
Lepidoptera, and Acari (mites) (Ebbert, 1991 & 1993)
 Includes :
1) Spiroplasmas
2) L-Form Bacteria
3) Wolbachia

32. Spiroplasmas
 Spiroplasmas - maternally inherited, transovarially
transmitted and lethal to male embryos
 Specific virus associated with each spiroplasma
 Transmitted between species by injecting
hemolymph
Eg. The sex ratio condition of Drosophila willistoni and
related neotropical willistoni group species is due to
Spiroplasmas

33. L-Form Bacteria
 Have a benign relationship with its own host
• Can be transferred through the egg cytoplasm
• Can be microinjected into females and can produce
expected male sterility
Eg. Drosophila paulistorum – associated with
Streptococcal L-form bacteria

34. Wolbachia spp.
 Most common cytoplasmically inherited m/os in
arthropods
 Obligate, gram-negative “rickettsial-like bacteria”
• Present in 17 to 76% of all arthropod species
 Causes the following in arthropods :
1) Thelytoky (only females)
2) Male killing
3) Cytoplasmic incompatibility (CI) - important
4) Female mortality

35. Cytoplasmic Incompatibility (CI)
 Occurs when Wolbachia-infected males mate with
uninfected females - resulting in a failure to produce
progeny in diplo-diploid species
• Wolbachia causes thelytoky in Hymenoptera -
Aphelinidae, Encyrtidae, Eulophidae, Pteromalidae,
Torymidae, Trichogrammatidae, Cynipidae, Eucoilidae,
Braconidae, Ichneumonidae, Proctotrupoidae
• Hypothesis : Wolbachia infections causing
thelytoky as a mechanism for the process of
speciation

36. A Mite Consisting Only of Haploid Females
 False spider mite, Brevipalpus phoenicis (Acari:
Tenuipalpidae) – consist entirely of females that
have only a haploid chromosome set (Weeks et al.,
2001)
 Due to the presence of an endosymbiotic bacterium
(not Wolbachia) which has feminized haploid males
• If female mites are treated with antibiotics, about
half their progeny become male

37. Male Killing endosymbionts in Coccinellidae
 Male-killing endosymbionts - Rickettsia,
Spiroplasma, Flavobacteria and Wolbachia –
association
 Coccinellids are susceptible to invasion by and
establishment of male-killing microbes because of their
biology
 Male killing evolved due to vertical transmission of
the bacteria from mother to eggs (transovarial)

38. Advantages of Sex determination
 Resolving the molecular genetics of sex
determination in arthropods and learning how to
modify sex ratio or fertility have both theoretical and
applied applications
 It can lead to improved genetic control of pests or
useful genetic modifications of beneficial biological
control agents

39. Applications of Sex Determination
 Genetic Control – eg. Eradication of screwworm
(Cochliomyia hominivorax) from North America
 “Sterile insect release method” (SIRM) or “Sterile
insect technique” (SIT)
 Involves mass-rearing and sterilization of males by
chemicals or irradiation and subsequent release to
mate with wild females
 Females mate only once, so any wild female
mating with a sterile male fails to contribute
progeny to the next generation (Knipling, 1955)

40. Applications of Sex Determination
 “Sterile insect release method” (SIRM) or “Sterile
insect technique” (SIT)
 Also applicable in : Ceratitis capitata, Glossina
palpalis, G. morsitans, Anopheles albimanus, Cydia
pomonella and ticks (LaChance, 1979)
 Genetic control - safe, specific, limited negative
impact on the environment – alternative to chemical
control