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ESPM 144/Insect Physiology/Prof. Mark Tanouye LECTURE 3: THE MALE REPRODUCTIVE SYSTEM AND SPERMATOGENESIS READING: Klowden pp.119-125 A. LAST TIME: looked at mating behavior, courtship (slide). Major features: aggregation (males, females finding each other)(hunting, calling, swarming, territorial), courtship, copulation, courtship mutants, mosaics. B. SIGNIFICANCE OF SEXUAL REPRODUCTION: (slide reminder) Genetic effect: create new diversity in the diploid stage at each generation. During gametogenesis, recombination (crossing over) new combinations of genes on same chromosome. By shuffling genes, recombination allows favorable and unfavorable mutations to be separated and tested as individual units in new assortments. Recombination occurs at 4n stage. (slide: spermatogenesis- genetic) terminology: spermatogonium (2n progenitor of primary spermatocyte), primary spermatocyte (2n), secondary spermatocyte (2n), spermatid (1n), haploid cell, develops into spermatozoon w/o division, process called spermiogenesis), spermatozoon (1n fertilizing gamete). C. CELLULAR DESCRIPTION OF SPERM: (generalized insect spermatozoon slide). Head region, mostly nucleus, DNA arranged in strands parallel to long axis. Front=acrosome: attachment of sperm to egg; maybe lysis of egg membrane. Axial filament (or axoneme): thought to produce undulating movements used in sperm locomotion. (variations in sperm slide). Often 300 um long x 1 um dia. Drosophila sperm 1.7 mm long. D. OVERVIEW OF MALE REPRODUCTIVE SYSTEM: (flow diagram slide): 3 major things: production, storage, delivery=testis, seminal vesicle, penis. Other things: ducting system: vas deferens (to seminal vesicle), vas efferens (away from testis) (vas=duct, vessel) (testis to seminal vesicle); ejaculatory duct (seminal vesicle to penis). One or more accessory glands provide secretions. (male reproductive system slide): (variations slide). E. MALE TESTIS: made up of testis tubes or follicles (Coleoptera Adephaga 1 follicle, lice 2 follicles, Acrididae over 100). (Drosophila??). Diptera simple undivided sacs (regarded as single follicle). F. DROSOPHILA SPERMATOGENESIS: (summary slide). Stem cell division (10 h), cyst development (250 h) therefore, steady-state 25 developing cysts (1 family) from each stem cell. Each testis 5-8 families of 25 developing cysts. Other details: 2 cyst cells form envelope around cyst (head cyst cell, tail cyst cell). 4 mitotic divisions (50 h) give rise to 16 primary spermatocytes (cytokinesis incomplete so all 16 interconnected). Growth phase (90 h) undergo 25-fold increase in volume. 2 rapid meiotic divisions (64 spermatids, incomplete cytokinesis, interconnected by ring canal). Spermiogenesis (134 h). Elongation part takes 35 h=50 um/h. (testis follicle slide). Some variations: number of mitotic divisions (eg. Melanoplus, 7 divisions, 512 sperm/cyst), degeneration (eg. many coccids, 2 sperm/spermatocyte, 32 sperm/cyst). G. TRANSFER OF SPERM TO SEMINAL VESICLES. By peristalsis (sperm not motile). (Accessory gland secretions slide) E. SPERMATOPHORE (slide). Primitive method of insemination. Gelatinous capsule which surrounds sperm. Not all insects (Apterygota, Orthoptera, Dictyoptera, Heteroptera, Neuroptera, some: Trichoptera, Lepidoptera, Hymenoptera, Coleoptera, very few Diptera). 1) spermatophore on the ground, 2) spermatophore passed directly to the female. F. INTERNAL FERTILIZATION (some orders of Heteroptera, Mecoptera, Trichoptera, Hymenoptera, Coleoptera, and Diptera) stream of fluid from accessory glands driven along ejaculatory duct and into the female by contractions of the glands; sperm injected into the stream by contractions of seminal vesicle (Aedes aegypti, Drosophila). G. IN FEMALE, SPERM STORED IN SPERMATHECA for future fertilization of eggs. F. STERILIZATION AND MUTAGENESIS: treat males with X-rays in order to introduce defects into sperm, for the purposes of sterilization (biological control, sterile releases), for the purposes of mutagenesis (Drosophila genetics, generation of new mutants). (electromagnetic spectrum slide) visible spectrum, long ultraviolet, short ultraviolet, X-ray, gamma ray. Key: energy present in different wavelengths of the spectrum. Study of vision (later): photoreceptor cells can utilize because of specialized molecules tuned to the relatively low energies present in visible spectrum (even long UV). Problem with short wavelengths: energy level is too high. Absorbed by all macromolecules (proteins, DNA). In this high energy state, bonds in the macromolecule break. Breaks in DNA, basis of mutagenesis, sterilization. (ozone layer slide). Life on earth possible only after ozone layer, cut short wavelength spectra down to acceptable levels. Macromolecules stable. Repair mechanisms. Mutations from mistakes in repair. Sterilization from so much damage that can't be repaired. (sperm age table + brood ages slide). (sex-linked lethal, T2;3s slide) explain sex linked. Also linear vs exponential.

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ESPM 144/Insect Physiology/Prof. Mark Tanouye LECTURE 4. THE FEMALE REPRODUCTIVE SYSTEM AND OOGENESIS READING: Klowden pp. 109-122 A. FEMALE REPRODUCTIVE SYSTEM (overview slide). Sperm reception and storage, egg production and laying. Vagina (genital chamber, bursa copulatrix, opening=vulva), spermatheca, ovaries, ovipositor. Also ducting system (main=oviduct), accessory glands. (generalized reproductive system slide, variations slide). Eggs fertilized in oviduct just before laying. B. SPERM RECEPTION, STORAGE. Receives sperm, direct insemination or spermatophore. Transfer to spermatheca: probably mostly passive (packed and transferred in packets called spermatodesms), some like to think sperm actively move to spermatheca guided by chemical stimulus (not too much evidence). Some insects spermatophore (eg. Rhodnius) placed in bursa copulatrix; transfer of sperm by contractions of female ducts. Component from male accessory gland initiates contractions. C. FERTILIZATION. In spermatheca, spermatodesms break down. Sperm become active, nourished by secretions of female accessory glands (actually spermatheca gland). As each egg passes down oviduct, a few of the stored sperm released from spermatheca enter oviduct and fertilize egg. Don't really know how this happens (ideas: compressor muscles of spermatheca, sudden pulses of hemolymph pressure, sperm swim out of spermatheca (Nasonia) after activation by pH change). D. THE OVARY. Composed of ovarioles (egg tubes)(see repro variations slide) (SEM slide). (Number of ovarioles slide). (Types of ovariole slide) panoistic (primitive), meroistic (telotrophic) (polytrophic). E. OOGENESIS, polytrophic example, most complicated. (Drosophila oogenesis slide). Probably only one stem cell per ovariole. 2 eggs per ovariole per day, therefore, about a 12 h production time. Follicle also called cyst. One oocyte, 15 nurse cells attached together because of incomplete cytokinesis (slide of 8 cell stage). Consistent pattern: 2 cells (one is oocyte) have 4 bridges; 2 cells have 3 bridges; 4 cells have 2 bridges; 8 cells have one bridge. Function of intercellular bridges: passage of cytoplasmic organelles and molecules between cells. Determination of oocyte, not clear. Needs 4 bridges. Most central location in cyst?? (species variation slide)(differencebetween panoistic, meroistic slide). Trophocytes initially bigger than oocyte (become polyploid): supply massive amounts of RNA; rRNA, tRNA (i.e. almost all of the RNA for the oocyte). Movement of material thought to be active, along electrical potential gradient. Subsequently, trophocytes collapse cytoplasm, organelles, sometimes nuclei go into oocytes. Meiosis after the 16 cell stage. F. VITELLOGENESIS: (slide). Vitellogenin=yolk. Synthesized (but not accumulated) in the fat body. Released into hemolymph until where they remain until uptake. Drosophila yolk proteins resolved into three peptides (MW about 46,000). Hyalophora (Lepitoptera) proteins in female not male. Absorbed preferentially by oocyte (20-30X more concentrated in oocyte than hemolymph). 1) Follicle epithelium retracts from the surface of the oocyte; 2) gaps appear between follicle cells thereby giving free access of hemolymph to surface of the oocyte; 3) proteing concentrated at oocyte membrane (mechanism unclear). Other things: vitelline membrane form on the outer layer of oocytes: some cases modification of oocyte plasma membrane; other cases forms in intercellular space between oocyte and follicle cells with droplets of material contributed largely by follicle cells. Egg shell (chorion, endochorion, exochorion): formed by follicle cells. For Rhodnius: ENDOCHORION: 1) polyphenol layer; 2) protein layer (becomes tanned); 3) second polyphenol layer; 4) amber-colored layer (oil + tanned protein); 5) second protein layer. EXOCHORION: 1) soft lipoprotein layer; 2) resistant lipoprotein layer. G. MATURE OOCYTE (an egg slide, various forms of eggs slide). Chromosomes arrested at metaphase I of meiosis. Completion of meiosis initiated by sperm entry. Ovulation, passage of oocyte into oviduct.. F. 2) 3) 4) 5) 6) 7)

ACCESSORY GLANDS. 1) produce glue for egg-sticking to substrate, Produce ootheca (egg-laying case, cuticle-like structure, in Periplaneta) Egg pools of grasshoppers, gelatinous sheath of Chironomus (Diptera) Egg cocoon (made of silk, in Hydroplulus (Coleoptera). Poison glands (Hymenoptera) Ovipositor lubrication Ant pheromones

H. OVIPOSITING. I. FEMALE STERILE MUTANTS. 650 loci give female sterility.

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INSECT SEX

parental

MALE

DIPLOID

FEMALE AGGREGATION

gametogenesis meiosis recombination

[COURTSHIP]

HAPLOID

COUPLING

MEIOSIS

gamete

fertilization

COPULATION FERTILIZATION

SIGNIFICANCE OF SEXUAL REPRODUCTION

PROGENY

DIPLOID

progeny

SPERMATOGENESIS

(general)

INSECT SPERM (idealized)

DIVERSITY IN ARTHROPOD SPERM

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MALE REPRODUCTIVE SYSTEM (overview) SPERM

PRODUCTION

testis

(idealized)

STORAGE

seminal vesicle

DELIVERY

penis

DUCTING (vas deferens, ejaculatory duct)

MALE REPRODUCTIVE SYSTEM

ACCESSORY GLANDS

TESTIS FOLLICLE MALE REPRODUCTIVE SYSTEM (examples)

DROSOPHILA SPERMATOGENESIS stem

10hr

stem

FAMILY OF 25 DEVELOPING CYSTS

primary spermatogonium

ONE CYST

4 mitotic divisions + 2 meiotic division

64 SPERMATOZOA 250hr

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SPERMATOGENESIS IN MOTH (during larva and pupal stages)

ACCESSORY GLANDS white gland opalescent gland hyaline gland

SECRETIONS 1.  2.  3.  4.  5.  6. 

Seminal Fluid (fluid matrix) Nutrients for sperm Stimulants for sperm motility Nutrients for developing egg (in female) Inhibitors of female receptivity Production of Spermatophore

ELECTROMAGNETIC SPECTRUM

SPERMATOPHORES

wavelength (cm)

ENERGY =

104 103 102 101 100 10-1 10-2 10-3 10-4 10-5 10-6 10-7 10-8 10-9 10-10 10-11

hc wavelength

h = Planck's constant c = velocity of light

STERILITY

RADIATION EFFECTS ON DROSOPHILA SPERM LETHAL MUTATIONS

(1cm) (1mm)

(1µ)

(1Å)

INSECT SEX MALE

FEMALE AGGREGATION

[COURTSHIP] COUPLING COPULATION FERTILIZATION

PROGENY

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FEMALE REPRODUCTIVE SYSTEM vagina SPERM RECEPTION

GENERALIZED FEMALE REPRODUCTIVE SYSTEM

ovary EGG PRODUCTION

SPERM STORAGE spermatheca

EGG LAYING ovipositor

FEMALE VARIATIONS

DROSOPHILA OVARY OVARIOLE OVARY

VARIATIONS - OVARIOLE NUMBER Locust Grasshopper Calliphora Drosophila

116 ovarioles 8 200 20-60

Termite Queen

4,000

Melophagus Hippobosca Glossina

4 4 4

most Lepidoptera

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OVARIOLE TYPES PANOISTIC (PRIMITIVE) Thysanura, Odonata, Plecoptera, Orthoptera, Isoptera

MEROISTIC - TELOTROPHIC TYPE Heteroptera, some Coleoptera

MEROISTIC - POLYTROPHIC TYPE some Coleoptera, Hymenoptera, Lepidoptera, Diptera

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DROSOPHILA OOGENESIS stem

stem

OOGONIUM

8-cell stage

(4 mitotic divisions) 1,500 follicle cells ONE OOCYTE + 15 NURSE CELLS (trophocytes)

OVARIOLE TYPES

FOLLICLE

SPECIES VARIATIONS

MEROISTIC TELOTROPHIC POLYTROPHIC

PANOISTIC

TROPHOCYTES PER FOLLICLE Dermaptera Aedes, Melphagus Dytiscus (Coleoptera) Apis, Bombus Carabus (Coleoptera)

1 7 15 48 127

NUMBER OF FOLLICLES/OVARIOLE Schistocerca Drosophila Oncopeltus Melophagus

20 6 8 1

FEMALE ACCESSORY GLANDS 1.  2.  3.  4.  5.  6.  7.  8. 

Produce Glue (for egg-sticking to substrate) Produce Ootheca (egg-laying case), Periplaneta Egg pools of grasshoppers Gelatinous sheath - Chironomus (Diptera) Egg cocoon - silk Hydroplulus (Coleoptera) Poison glands (Hymenoptera) Ovipositor lubrication Ant pheromones

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