Genetic Systems, Genome Evolution, and of Development

Chapter Four

Genetic Systems, Genome Evolution, and

Genetic Control of Embryonic Development

Genetic Systems Insects are very diverse and ancient

1/2 of all described species

Perhaps 75 % of all animals

~ 883,475 spp. in 762 families in 32 orders now

Diverse life styles and genetic systems

Genetic Systems Most insects are diploid (2 n) in soma and haploid

(n) in gametes Some are parthenogenetic Some are polyploid

Parthenogenesis: 3 main types Arrhenotoky: males haploid, females diploid Thelytoky: all females Deuterotoky: unfertilized eggs –> either male or female

(rare)

Genetic Systems Thelytoky

Arisen repeatedly, several types

Sole mode or alternate with sexual reproduction

Sometimes produced by chemical or physical stimuli

Occurs spontaneously in many spp. at low rate

Genetic Systems Parahaploidy (mealybugs)

Fertilized eggs lose chromosomes derived from father –> haploid embryo, then becomes a male

This system must involve some sort of marking (imprinting) of paternally derived chromosomes

Genetic Systems Endopolyploidy found in many cells

Ploidy is a difficult topic ( polyteny vs polyploidy )

Many insects have one or more polyploid tissues (multiple copies of the chromosomes)

Ex: haploid male bees have same amount of DNA as females because cells are endopolyploid

Genetic Systems Much of what we know about insect genetics is

based on Drosophila melanogaster Complete genomes of several Drosophila

species available Other genomes have been sequenced including: Anopheles gambiae, Aedes aegypti Apis mellifera

Bombyx mori Mediterranean fruit fly (Ceratitus capitata)

Tribolium castaneum

Genomes Sequenced

Nasonia parasitoids Tsetse, Glossina morsitans Screwworm, Cochliomyia hominivorax Acyrthosiphon pisum, pea aphid Bombyx mori Solenopsis invicta Pediculus humanus Danaus plexippus, Monarch butterfly Tetranychus urticae, two-spotted spider mite Ixodes ricinus Metaseiulus occidentalis Plus more on an almost daily basis

Dynamic Genetic Systems Polyteny, polyploidy, gene amplification and other

unusual DNAs are found in different tissues at different stages Closed covalent circular DNAs in

D. melanogaster cell cultures: much repetitive; function unknown

Minichromosomes in D. melanogaster from TE1

Centromere - like elements in phorid fly

Horizontal Gene Transfer from Microorganisms to Insect Genomes

Examples include: Carotenoid-production genes from fungi in aphids

(both Aphis pisum and Myzus persicae)

Transfer of endosymbiont genes to arthropod genomes relatively more common than from free- living microbes

Bean beetle, Callosobruchus chinensis has ~ 30% of the Wolbachia genome in the X chromosome, function?

Horizontal Gene Transfer from Microorganisms to Insect Genomes

Examples include: Nasonia vitripennis genome has 13 proteins found in

poxviruses, probably introduced in Wolbachia Genes duplicated and are transcribed

Acrythosiphon pisum, the pea aphid, has 12 genes or gene fragments, most from bacteria other than its symbiont Buchnera

The coffee berry borer, has a mannase gene from a Bacillus bacterium that hydrolyzes the major storage polysaccharide in the coffee bean

B Chromosomes May not segregate normally in mitosis or

meiosis E.g., diversity found, significance not resolved

Discard notion that insect genomes are strictly

nuclear and mitochondrial

Germ - line Limited Chromosomes

During embryonic development in some insects, some chromosomes are lost These will become somatic cells Cells with full chromosome complement are

germ - line cells

Unique - Sequence DNA

Most genetic information is unique Proportion of unique sequences varies among species (55 to

80 %) Genes are present in multiple copies in some cells due to

polyploidy or gene amplification Gene amplification: a portion of chromosome is replicated

Ex: chorion genes, some pesticide R genes

Gene Amplification: the onion-skin model

Middle - Repetitive Nuclear DNA Found in more than one copy, but modest

amounts ribosomal RNAs (rRNA) transfer RNAs (tRNA) (90 found in Drosophila

encoded by at least 670 genes) histones, actins, cuticle, hsp, larval

serum, silk, vitellogenin genes Allows large amounts of gene product to be

produced in a short time in a coordinated manner Often present in tandem arrays

Middle - Repetitive Nuclear DNA Heat - shock response initially discovered in

D. melanogaster

Universal from bacteria to man 9 chromosomal sites puff after heatshock 7 heatshock proteins produced; including

hsp70, hsp83, small heat - shock genes 10 copies of hsp70 in D. melanogaster;

most abundant and highly conserved Act as molecular chaperones

Middle - Repetitive Nuclear DNA

Histone genes: 5 histones in chromosomes Share regulatory sequences, are coordinately expressed Typically lack introns In tandem array Copy number varies among Drosophila species Variability due to location? Eu - vs. hetero- chromatic regions


Immune response: Protecting against bacteria, viruses, fungi, parasitoids with cellular and humoral immune responses

Constitutive and inducible responses Antibacterial proteins or peptide families:

cecropins, attacins, lysozmes, defensins Families often tightly clustered

Middle - Repetitive Nuclear DNA Ribosomal genes: Ribosomes have 2

subunits, each of RNAs and proteins

Number of ribosomal genes varies D. erecta has 160 while D. hydei has > 500 Most insects have 200 to 500 Two clusters in Drosophila: transcribed as

a single unit – an efficient method


Silk genes: silk in cocoons, egg stalks, capture nets

Composed of fibroins, which consist of several aa sequences in reiterated arrays

Silk gland of Bombyx mori is polyploid (20X) and large amounts can be produced in 5 to 6 days

Fibroin and sericin –> silk

Middle - Repetitive Nuclear DNA Yolk protein (vitellogenin) genes: food for

embryos Drosophila -- 3 polypeptides: YP1 expressed in fat

body and secreted into hemolymph to be delivered to oocytes

Production and expression of YP1-3 coordinately regulated and under control of 2 hormones (JH and ecdysone)

Production rate is high (1/3 total hemolymph proteins): only one small intron in YP1, YP2

YP1 and YP2 on X chromosome; YP3 more distant on X, due to gene duplication ?

Middle - Repetitive Nuclear DNA Transposable elements: DNA sequences that can

move to new sites: 2 main classes Can invert, undergo deletion or amplification

Class I: related to retroviruses that have long terminal repeats (LTRs) Transpose by reverse transcription of an RNA

intermediate Also includes elements with no LTRs: non-LTR

retrotransposons


Transposable elements: Class II: Transpose directly from DNA to DNA

Includes elements with short inverted terminal repeats and have a coding region for a transposase

NEW class: rolling - circle transposons or Helitrons Found in Eukaryotes (including insects) Replicate by a rolling-circle method


Transposble elements are significant Many TE types found in Insects: see tables for

names and descriptions

At least 1/2 of all spontaneous mutations in D. melanogaster due to insertions of TEs –> mutations, deletions, inversions, translocations

All characterized HIGHLY UNSTABLE genes in D. melanogaster contain a TE

Middle - Repetitive Nuclear DNA Origin and history of TEs

Might originate in a species or be acquired by

horizontal ( or lateral ) transfer (HT)

mariner found in most insects and some mites; most degenerated and inactive

Evolutionary history of arthropods and mariner NOT CONGRUENT –> HT

Frequency of HT unknown, but has implications for evolutionary theory and for risk assessment of transgenic arthropods modified using TE vectors

Middle - Repetitive Nuclear DNA HT may occur within viruses infecting

arthropods and other hosts

4 families of TEs were found in Rhodnius, which feeds on opossums, squirrel monkeys and other vertebrates.

The TEs in the insect and the vertebrates were similar, suggesting host-parasite interactions are important in HT

Highly - Repetitive Nuclear DNA If has uniform nt composition, it can

separate out on centrifugation as satellite DNA Mini or micro satellite DNA depends on

length: Micro = 1 to 6 bp

May be a large amount of total DNA: 30 to 70%

Role in evolution not understood (some function as telomeres and centromeres)

High Rates of Protein Production

Achieved several ways Duplication of chromatids –> polyteny, polyploidy

Hypertranscription

Gene amplification: replication of a gene at a single locus so multiple copies can be transcribed at once

Gene duplication: copying a gene and maintaining it on same chromosome in tandem array or on other chromosomes

High Rates of Protein Production

Drosophila: chorion relatively simple, endo- and exo-chorion of 6 major and 14 minor proteins produced over 5 hr Gene amplification results in large amounts of proteins in 2 chorion gene clusters, one on X and one on III

Clusters 5 to 10 kb in size, encoding tandemly oriented chorion genes

Amplification is ca. 20 - fold on X and 80 - fold on III –> �onion – skin� structure

Amplification of Drosophila Chorion Genes 3 characterized chorion genes in this cluster; polarity of 4th gene not resolved

Silk Moth Chorion Genes

Solved rapid production problem by gene DUPLICATION

Multiple copies of divergently transcribed, coordinately expressed genes Ex: Two late gene families in 15 pairs on a

140 - kb segment

Homology maintained by concerted evolution

Insecticide Resistance and Gene Amplification

Amplification of esterase genes in the aphid Myzus persicae and the mosquito Culex pipiens result in identical gene copies present in tandem arrays

Myzus persicae resistant to neonicotinoids by gene amplification of a single P450 gene

Because exposure to toxins can induce mutations in cell

cultures, is it possible that some insecticide R genes are CAUSED by pesticide use (rather than being �preadaptive mutations� waiting to be selected on)

Multiple Genomes in Insects

Nucleus, mitochondria, multiple microbial symbionts viruses, bacteria, fungi, protozoa

WHAT is the individual?

Symbionts are intra- and extra - cellular

In gut and reproductive tract, elsewhere

Rice weevil, Sitophilus oryzae, has 4 genomes: nuclear, mt, principal endosymbiont, Wolbachia


Symbionts: may provide metabolic products for hosts

Obligatory vs. facultative

Have specialized structures

Often transmitted in specialized manner (often transovarial)

Many difficult to study if cannot be cultured

Symbionts and Insects

Symbiosis is a broad term, including parasites, pathogens and mutualistic interactions

Symbionts may be Eubacteria, fungi, yeasts, viruses, protozoa or Archaea

Many microbes on the outside of insects are transient, but not all

Symbionts may provide nutrients, affect host range, temperature tolerance, longevity, fecundity, sex ratio, behavior, responses to natural enemies, etc.


Symbionts: may increase probability an insect vector can transmit disease

Rickettsia - like organisms in tsetse affect (produce endochitinases) sleeping sickness trypanosomes, reducing transmission rates

Separation of symbiont and self: host immune system affected ?


�Bug Within Bug�: A first

Mealybugs have endosymbionts in cytoplasm of polyploid host cells in bacteriomes –> nutrients to hosts

Relationship 100 to 250 million yr old

Endosymbionts surround host cell nucleus and consist of 2 bacteria: spheres are Β-proteobacteria with γ proteobacteria within the first bacterium


The bean bug Riptortus pedestris has a gut symbiont in the genus Burkholderia

The adult bug has up to 10-8 bacteria

The symbiont is transmitted in the soil If the bug picks up a strain that is R to a pesticide

(fenitrothion), the bugs become R

The R bacteria can be spread when the bug flies to new sites

Multiple Genomes in Arthropods

An unusual symbiont is the bacterium Candidatus Midichloria mitochondrii

Found in the mitochondria of hard ticks (Ixodidae)

The bacteria reduce the number of mitochondria and are transstadially transmitted

Function unknown: common in field, fewer in lab colonies

Wolbachia α - proteobacterium common in insects

Intracellular, gram - negative rods

Not readily cultured

Infect 17 to 76 % of all arthropods

Have diverse effects on hosts, including �none known�

Also in Crustacea and nematodes

Wolbachia

May alter sex ratio (thelytoky, male killing) and sex determination (Ch. 10) and cause cytoplasmic incompatibility

Due to ability of Wolbachia to modify sperm?

Eggs of females infected with same strain of Wolbachia are rescued but uninfected females –> dead embryos

Incompatibility partial or complete Incompatibility bi- or uni- directional

Microbial Symbionts Can Alter Biology of Hosts What is an individual?

Wolbachia May be only in germ line or in all tissues

Sometimes can be transferred to new populations by microinjecting egg cytoplasm into uninfected eggs

Wolbachia evolved 80 to 100 mya

Arthropod ancestor occurred at least 200 mya

Wolbachia invades arthropods through HORIZONTAL TRANSMISSION

Horizontal Transfer of Wolbachia

Much remains to be learned

Wolbachia may contain bacteriophages (WO)

WOs may move horizontally also

Phage may confer benefit on Wolbachia?

The Many Effects of Wolbachia

Block transmission of disease-causing agents Cytoplasmic incompatibility Male killing Modification of immune responses Parthenogenesis Nutritional mutualism Temperature effects

Cardinium

Relatively recently identified, less well studied

Can cause many similar effects as Wolbachia

Cytoplasmic incompatibility (spider mites, Encarsia species, Metaseiulus occidentalis )

Thelytoky in Encarsia parasitoids, Brevipalpus mites

Host-selection behavior modified in Encarsia

Polydnaviruses Braconidae and Ichneumonidae infected (2

groups, distinct biology)

DS circular DNA genomes are segmented

Campoletus sonorensis virus consists of 28 DNA molecules, ranging from 5.5 to 21 kb: total genome = 150 kb

Polydnaviruses enable parasitoids to parasitize hosts Replicate only in ovaries and secreted into

oviducts –> lepidopteran larvae

Polydnaviruses

Are vertically transmitted and integrated into chromosomes of wasp

Species - specific viruses

Polydnaviruses replicate asymptomatically in wasps but cause pathogenic infection in Lepidoptera Venom + virus –> full effect in some

Obligate - mutualistic association

Gut Symbionts

Many in guts; relatively little understood

Especially important in hind gut Hindguts of termites -- small bioreactors

with distinct microhabitats Termite guts contain Bacteria, Archaea,

Eukaryotes, Yeasts Molecular analyses indicate more species are

present than previously recognized

Cockroaches also have gut microbial communities, but are less interdependent.

Antlions and Salivary Gland Bacteria

Suck out fluids after paralyzing prey with a toxin

Toxin produced by bacteria in salivary glands Toxin a homolog of GroEL, a heat - shock

protein in E. coli

Will other insecticidal proteins from fluid - feeding predators be produced by other endosymbionts?

Tsetse and Symbionts

Vectors of sleeping sickness Microorganisms found in midgut, hemolymph, fat

body, ovaries Primary symbiont: Wigglesworthia glossinidia is

intracellular in U - shaped bacteriome in anterior gut

Secondary: Sodalis glossinidius in midgut Both transmitted in milk - gland secretions Wolbachia in reproductive tissues, transmitted

transovarially

Tsetse and Symbionts Efforts to eliminate symbionts –> retarded

growth and reduced reproduction

Difficult to eliminate only one symbiont with antibiotics, so difficult to resolve which does what

Gut symbionts –> supply B - complex vitamins Sodalis produces a chitinase, which increases

transmission of sleeping sickness agent Evolutionary analyses suggest W infections came first, then S No evidence for horizontal transfer between tsetse

species

Rhagoletis pomonella Symbionts

Apple maggots contain Enterobacteriaceae in the gut and female reproductive organs

In addition, Klebsiella oxytoca is found in the gut Both types are abundant in esophageal bulb, crop and midgut forming a biofilm

Symbiosis in Fungus-growing Attine Ants Attine ants live in tropics, carry leaf fragments to nests where they fertilize a fungus, which is their food

The fungi produce specialized structures that are

consumed by the ants and the workers maintain the gardens as well as care for the brood

New queens carry the fungus within a pouch in her oral

cavity to new nest sites The ant-fungus relationship is complex

Symbiosis in Fungus-growing Attine Ants The food fungi are attacked by a microfungus

(Escovopsis) An actinobacterium (Pseudonocardia) produces

antibiotics that inhibit the Escovopsis A black yeast (Phialophora) parasitizes the ant-

actinobacteria mutualism The symbiosis may be at least 50-60 million years old

Symbiosis in Fungus-growing Attine Ants Ant metapleural glands produce antimicrobial compounds that help protect ants from the insect-pathogenic fungi

The �waxy exudate� on the body are aggregations of the actinobacteria (Pseudocardia) growing in crypts with glands under the crypts that produce secretions used by the actinobacteria

The exudate protects the fungal food

Symbiosis in Fungus-growing Attine Ants

Ant with foveal openings on body These crypts contain bacteria that produce antibiotics that protect the fungal food Left: light transmission showing dense bacteria Right: Transmission EM showing single cell with bacteria within the crypt

Symbiosis in Southern Pine Beetles

Dendroctonus frontalis have a mutualism similar to attine ants Adults carry a beneficial fungus in a special storage compartment called a mycangium Females excavate galleries in the inner bark and phloem of pines to oviposit and inoculate galleries with the beneficial fungus that is food for their progeny A fungus that can out compete the �food� fungus can affect the relationship

Aphids and Buchnera

Well studied endosymbiont: Mutual benefits

Complete genome sequenced Gut symbionts –> in bacteriocytes –> supply

hosts with aa Aphids become sterile or die if symbionts

eliminated Relationship stable for ca. 250 million yrs About 9% of Buchnera genome devoted to

producing essential aa for aphids Genes for nonessential aa absent in

Buchnera: symbiont DEPENDS ON HOST

Aphids and Buchnera

Vertical transmission has occurred

Co - speciation of aphids and Buchnera

Tryptophan and leucine genes on plasmids in Buchnera, which allows increased expression

Plasmid copy number varies in species

Genome reduced to about 650 kb, about one-seventh size of E. coli

Buchnera has 50 to 200 chromosomes, no. varies with host stage

Aphids and Other Symbionts

Facultative symbionts occur in different populations

Pea aphid:

Protect aphids from entomopathogenic fungi and parasitoid wasps

Enhance temperature tolerance

Change body color from red to green, possibly reducing predation by lady beetles

Another allows the pea aphid to feed on clover, broadening the host range

Insect Development Much learned from D. melanogaster

Molecular tools allow dissection of development

Embryonic development well studied

Important to understand when microinjecting to transform flies (discussed later)

Example of coordinated gene regulation

Insect Development

Embryogenesis

Fertilization initiates completion of meiosis I and II

Pronuclei fuse (syngamy)

Early cell division rapid so no cell growth occurs

Initial mitoses atypical because first 9 divisions result in a syncytium containing ca. 512 nuclei lacking cellular membranes

Embryonic Development in D. melanogaster

From fertilization to just before gastrulation

Nuclei migrate to periphery

Pole cells formed

Insect Development

Embryogenesis

Pole cells, which will develop into the germ line, develop around division 9

Finally, membrane invaginates to enclose each nucleus –> cellularized blastoderm

Cellular blastoderm completely surrounds internal yolk mass

After this stage, specific body segments are determined

Insect Development

Postembryonic Development

D. melanogaster is holometabolous

Sequential life stages with molts between each

Adult structures develop from cells in imaginal discs

Imaginal discs in larvae give rise to adult tissues

Holometabolus Life Cycle of D. melanogaster

Insect Development Dissecting development with mutants

Mutants allow geneticists to identify particular pathways

Systematic program of mutagenesis led to Nobel

prize for Nusslein - Volhard, Wieschaus and Lewis� work in 1995

Embryonic Development

Two main phases in D. m. embryos

1) Many genes encode transcription factors or nuclear proteins –> cascade of transcriptional factors regulating other genes

Results in successive division into smaller and smaller domains by differential and combinatorial action of transcription factors

Ends at time of cellular blastoderm


Two main phases in D. m. Embryos

2) Begins after cellular blastoderm and elaborates on information provided from reference points deposited along dorsal - ventral and anterior - posterior axes.

Requires transfer of information between cells

Homeotic describes replacement of one part of the body by a

serially homologous part

Homeotic mutations yield four - winged D. melanogaster


Three gene classes control embryonic development in D. melanogaster

1) Maternal - effect genes specify egg polarity and spatial coordinates of egg and future embryo

2) Segmentation genes (gap, pair-rule and segment polarity classes) determine number and polarity of body segments

3) Homeotic genes determine identify of segments

Development of segments in embryos involves a hierarchy of regulatory genes Maternal - effect genes are first Zygotic genes are next


Maternal - effect genes

Important in development of egg to blastoderm

Affect life histories of insects, including incidence and intensity of diapause

wing polyphenism dispersal development time resistance to chemicals and microbial infection


Zygotic Segmentation Genes

Three classes: pair - rule gap segment polarity Apparent segments not valid: parasegments 14 parasegments in D. melanogaster


Gap Genes

Named because large areas of normal cuticular pattern deleted in mutants

Ex: Kruppel, hunchback, knirps Contain DNA-binding domains

Segmentation Gene Classes Illustrated by Mutations

Interactions During Development

Normal development requires coordinated expression of thousands of structural genes in a controlled manner

Controlling genes presumably arranged hierarchically or form a network to ensure proper timing

Development in Other Insects

Tribolium development analyzed

Gene order of 6 homeotic genes in single cluster is homologous to Antennapedia and bithorax complexes of Drosophila

Homeotic Mutations in Tribolium

maxillopedia wild type

wild type

Homeotic Mutations in Tribolium

cephalothorax results in incorporation of prothorax into head and labial palps into antennae

Development in Other Insects

Comparative studies on evolution of developmental genes in insects may provide understanding of basic mechanisms of genetic control of development

Evo - Devo

New discipline: evolutionary developmental biology emerged recently Combines comparative embryology paleontology molecular phylogenetics genome analysis

Evo - Devo

Goals include understanding: Origin and evolution of embryonic development How modifications of development lead to

novel features Adaptive plasticity of development in life -

history evolution How ecology affects development to

modulate evolutionary change Developmental basis of parallel evolution

and homology

Evo - Devo

Homology: Difficult and �fuzzy� term At least 9 concepts in comparative biology

literature Because genes play multiple roles in

development, it is difficult to resolve homologies

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Genetic Systems, Genome Evolution, and of Development