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Among animals, differences between
the sexes are usually specified by differential gene activities in individuals
that are genetically determined to be either males or females. The
sex-specific information is given by a primary sex-determining signal at the
beginning of a biochemical surge that results in development of a male or
female individual. Primary sex -determining signals vary among animal groups
(Bull 1983). Thus, the primary sex determining signal in humans and
butterflies is the identity of the sex chromosomes (XX, XY for humans and ZZ,
ZW for butterflies), and the primary sex determining signal for the fruitfly Drosophila
melanogaster, and the nematode Caenofhabditis elegans, is the
ratio of sex chromosomes to other chromosomes. Few of the primary genetic
signals of sex determination have been dissected to their molecular and
genetic basis for only a few organisms, all of which have sex chromosomes.
However, not much information exists for organisms that do not have sex
chromosomes. Sex Determination in the
Hymenoptera Hymenoptera demonstrate a
different mode of sex determination.
Here, males develop nom unfertilized; haploid eggs, and females
develop from fertilized eggs that are diploid. This kind of sex determination
is known as haplo-diploidy, and it is understood at the chromosomal, but not
the molecular level, in a number of species of Hymenoptera. A number of
hymenopterans share a mode of chromosomal sex determination known as
'complementary sex determination' (CSD) (Cook & Crozier l995; Wu et al.
2003). There are two basic types of CS: single locus CSD and multiple loci CSD.
Under single-locus CSD, sex is determined at one highly polymorphic genetic
locus known as the 'sex locus'. Fertilized eggs may be heterozygous at the
sex locus and develop into females, or homozygous and develop into diploid
males. Unfertilized eggs are hemizygous and develop into haploid males.
Diploid male production associated with CSD represents a strong genetic load
because diploid males are commonly inviable, sterile, or produce sterile
daughters (Godnay & Cook 1997; but see Cowan & Stahlhut 2004). The
severity of this genetic load increases with the frequency of inbreeding and
with decreased genetic diversity in general. Complementary sex determination
was determined in the parasitoid wasp Habrobracon hebetor in the
1940's using breeding studies and recessive eye color markers, which
identified paternal inheritance in males under conditions of inbreeding
(Whiting l943). CSD can cause severe shifts in sex ratio (toward males) as
well as declines in population growth because of the production of diploid
males, and can therefore reduce the effectiveness of parasitoids as
biological control agents (Stouthamer et al. 1992; Wu et al. 2003). However,
not all parasitoids have CSD, and Stouthamer et al. (1992) believed that
species lacking CSD are better equipped to control pest insects than species
that have CSD, a hypothesis that has received some empirical support (Heimpel
& Lundgren 2000). The genetic mechanism of sex
determination of parasitoids (or any hymenopterans) that do not exhibit CSD)
remains to be found, although genomic imprinting was implicated in
experiments on the parasitoid Nasonia vitripennis (Dobson &
Tanouye 1998). Even relatively closely related parasitoid species, however,
can differ in their mode of sex determination. In particular, a single
parasitoid genus (Cotesia) contains some species that do, and some
species that do not, exhibit CSD (Stouthamer et al. 1992; Niyibigira 2003a,b:
Gu & Dom 2003; De Boer et al. submitted). Studies on the honeybee, Apis
mellifera, have led to the discovery of a gene that acts as the primary
sex-determining signal (Beye el al. 2003; Beye 2004; Hassellman & Beye
2004). The discovery of this gene represents a major breakthrough in our
understanding of sex determination in the Hymenoptera and opens up the
possibility of understanding sex determination in other hymenopterans with
CSD, and also how it is that some hymenopterans can have CSD and others do
not. Sex locus linkage maps have been
produced from the honeybee (Beye et al.1994, 1996, 1998; Hunt & Page]
994; Hasselmann et al. 200];) and the parasitoid H. hebetor (Holloway
et al. 2000). In the honeybee, a relatively fine scale map was produced that
included a marker that flanked the putative sex locus by approximately 50 KB.
A chromosomal walk from this marker, along with positional cloning led to the
identification of a locus that was always heterozygous in females (Beye et
al. 2003). Molecular analysis of this region led to the discovery of 9 exons
spanning approximately 9 KB. Exons 2-9 produce the open reading frame of the
gene, which was named complementary sex determiner (csd) by Beye et al.
(2003) (Fig. 2). Two areas of particular interest were found in exons 6-9.
One is a domain that contains a series of repeated arginine (R) a serine (S)
amino acid. These repeats are characteristic for a family of proteins known
as SR proteins, which are known to playa role in mRNA binding in a number of
other organisms (Mount & Salz 2000; Beye et al. 2003). The highest degree
of homology to csd was found with the gene Iransfornler (Ira), which produces
an SR protein that is part of the sex-determining pathway in Drosophila
(Beye et al. 2003). In particular, Ira is involved in specific cleaving of
doublesex and fruitless rnRNAs, which results in expression of the female
phenotype in Drosophila (Cline & Meyer 1996). One major hypothesis
therefore holds that csd is a functional homologue of the Ira gene, and that
it serves a function in the biochemical sex-determination cascade comparable
to the function 'Of Ira in Drosophila (Beye 2004). Homologs of the Apis mellifera
csd gene have been found in A. dorsata and A. cerana (Cho et
aI, in press), and a 1 94-bp fragment of a csd-like mRNA of the apid Melipona
compressipes has been published on Genbank with 85% homology to the Apis
dorsata csd gene. Some of the
allele sequences from exons 2 and 3 (see Fig. 2) from A. dorsata and A.
cerana are more closely related to alleles from A. mellifera than
to other alleles from their own species (Cho et a). in press). This indicates
that some sex alleles are older than their species, a possible consequence
ancient polymorphism' and incomplete lineage sorting at these loci (Brower et
al. 1996). In the case of A. cerana and A. mellifera, molecular
clock analyses suggest that the two species diverged approximately 7 million
years ago, and that some of their sex alleles are 14 million years old. No
homology to csd has yet been found outside of the family Apidae as of 2006. As previously noted, CSD is
thought to be the ancestral mode of sex determination in the Hymenoptera. If
true, species that do not exhibit the CSD phenotype have somehow lost CSD but
retained haplo-diploidy. Alternative models of sex determination that are
compatible with haplo-diploidy include genomic imprinting, in which there is
differential expression of maternally and paternally inherited alleles for a
given gene or se1 of genes (Dobson & Tanouye 1998; McDonald et al. 2005),
genjc balance, where female- determining genes respond to the increased
dosage of DNA within a diploid cell and male- determining genes do not, and
multiple-locus (ml-CSD) (Cook 1993; Beukeboom 1995). ml-CSD was first
suggested by Crozier (1977) as a possible way to 'evolve away from' CSD. As
we explain below, we have evidence supporting a ml-CSD model for the
parasitoid Cotesia plutellae, and a more detailed investigation of
ml-CSD forms objective 2 of this proposal. As first put forth, diploid males
could only be produced under ml-CSD when all of 2 or more sex loci are
homozygous. This would decrease the production of diploid males with respect
to sl-CSD, even under conditions of inbreeding and genetic bottlenecks, and
could therefore greatly decrease the genetic load associated with CSD (Cook
1993a; De Boer et al. submitted; see below). Multiple-locus CSD could evolve
from sl-CSD by gene duplication. Gene duplication occurs often, either
through tandem duplication of the entire gene, segmental duplication of part
of a gene, or global duplication of the entire genome (Prince & Pickett
2002). Classical models predict that the loss of one redundant duplicate
should be the predicted evolutionary outcome, and that the retention of both
duplicates should happen far more rarely. However, retention appears to
happen more often than models predict (Prince & Pickett 2002). Duplicate
genes can be retained by changes in the protein-coding domain, or by changes
in the regulatory elements, leading to different spatial or temporal gene
expression. The first of these mechanisms (change in protein sequence) does
not seem to be a plausible explanation for ml-CSD because it commonly leads
to an entirely different function of the duplicated gene. A pathway by which
the retention of the duplicated gene becomes more likely was suggested by
Force et al. (1999) and is called the
duplication-degeneration-complementation model. This model is based on the
fact that most eukaryotic genes have more than one function. Each duplicate
gene then loses one or more sub-functions through degenerative mutations in
the regulatory sequences. If both duplicates need to be retained to be able
to cover the full function of the ancestral gene, they become complementary.
So instead of leading to new gene functions, gene duplication leads to
partitioning of ancestral gene functions. Indeed, gene duplication can
increase expression diversity and enable tissue or developmental
specialization to evolve (Liet al. 2005). Below, we discuss the implications
of gene duplication and ml-CSD on the construction of hypotheses for
mechanisms of CSD function. Contemporary Research In the haplo-diploid Hymenoptera,
unfertilized eggs develop as haploid males and fertilized eggs typically
develop as diploid females. In species that have single-locus complementary
sex determination (sl-CSD), fertilized eggs may develop as diploid males if
they are homozygous at a single locus (the sex locus). sl-CSD was discovered
in the 1940's by P.W. Whiting working in Habrobracon hebetor, and has
since been identified in over 50 species of hymenopterans, including
symphytans (sawflies), aculeates (ants, bees & wasps) and ichneumonoids
(braconid and ichneumonid parasitoids (Wilgenburg et al. 2006). Diploid males
are rare in nature because of the very high diversity of alleles at the sex
locus, but their frequency increases under inbreeding or genetic bottlenecks
(Cook & Crozier 1995). An exception is vespid Euodynerus foramilatus
(Cowan & Stahlhut 2004) where diploid males are developmentally inviable
or sterile and their appearance indicates a severe loss of fitness (Cook
& Crozier 1995). CSD is suspected to be a major impediment to successful
establishment of many exotic ichneumonoid parasitoids in classical biological
control because of the high risk of genetic bottlenecks inherent in the
process of biological control (Stouthamer et al. 1993; Heimpel & Lundgren
2000; Wu et al. 2003). Further insight of CSD resulted in
a greater understanding in recent years with the discovery and cloning of the
gene involved in sex determination under sl-CSD in the honeybee, Apis
mellifera, by Beye et al (2003) Beye (2004), Hassellman & Beye ( 2004). The gene has been called the
complementary sex determiner (csd) and interference with the csd transcript
converts genetic females into males (Beye et al. 2003). The existence of csd
should lead to a comprehensive understanding of the molecular pathways that
lead to sex determination in the honeybee. Further research by Heimpel &
associates revealed that sex determination in the parasitoid Cotesiaplutellae
(=C. vestalis) (Hymenoptera: Braconidae) is mediated by two sex
loci. Homozygosity at both loci is
probably required for production of diploid males in C. plulellae.
This mode of sex determination (multiple-locus CSD; ml-CSD) had been expected
as an extension of sl-CSD since the 1970's (Crozier 1977), but has not been
discovered until now by Heimpel & associates. " Would loss of CSD mean loss of csd
? Not all hymenopterans exhibit CSD. Hymenopterans
without CSD can inbreed for dozens of generations with no diploid male
production (e.g. Skinner & Werren 1980; Cook 1993a; Niyibigira et al.
2004a,b), have their genome duplicated by parthenogenesis-causing Wolbachia
without producing diploid males (e.g. Stouthamer & Kazmer 1994), or they
simply produce patterns of offspring sex ratio and mortality under modest
levels inbreeding that are incompatible with sl-CSD (e.g. Beukeboorn et al.
2000; Wu et al.2005). These species a]] achieve haplo-diploidy without CSD. A
viable alternative to CSD has been discovered in the continuous inbreeding
parasitoid, Nasonia vitripennis, which is one of the species for which
CSD had been previously ruled out. Dobson & Tanouye (J 998) used crosses taking advantage of a
supernumerary Chromosome (PSR for
'paternal sex ratio) that causes paternal genome loss in females to
provide evidence consistent with a
genomic imprinting model of sex determination. In their studies, female N.
vitripennis development depended upon the presence of chromosomes of
paternal origin, regardless of ploidy or heterozygosity. Whether or not genomic imprinting
turns out to be a general explanation for how sex is determined in hymenopterans
without CSD, the fate of the csd gene and the biochemical pathway that it
contributes to in hymenopterans that do not exhibit the CSD phenotype remains
unknown. The current state of knowledge regarding the distribution of CSD within the Hymenoptera can be summarized
as follows:: The CSD phenotype has been
described from over 50 hymenopterans from symphytans, acuJeates and
jchneumonoids, and the csd gene has been cloned and is under extensive study
in 3 species of Apis (Beye et
al. 2003; Cho et al, in press). Meanwhile, sl-CSD has been ruled out from
about] 8 species of hymenopterans, of whjch ml-CSD has also been ruled out
for 7 species. Most of the species that lack CSD belong to the large
hymenopteran clade called the 'Parasitjca' which has no members that do
exhibit CSD. However, species without
CSD are also found in the Aculeata
and the Ichneumonoidea, both of which have members with CSD.
Because of the phylogenetic distribution of the CSD phenotype, it has
been suggested that CSD is ancestral in the order, and that the loss of CSD
is an evolved condition that is favored evolutionarily because it achieves
haplo-diploidy without the production of diploid males (Cook & Crozier
]995; Godfray & Cook 1997). The absence of a CSD phenotype
does not preclude a role for the csd gene in sex determination. Csd shares
modest homology with transformer, a gene that is involved in the sex d
determination pathway of Drosophila
(Beye et aJ. 2003). In Hymenoptera that do not exhibit the CSD phenotype, two
thoughts can be articulated for the fate of the csd gene: (1) the csd gene may become deactivated
and cease to be transcribed and/or translated; (2) csd proteins may continue
to be produced and take part in the biochemical sex determination pathway,
but in such a way that heterozygosity is not needed for the production of
female offspring. These are the csd deactivation and csd incorporation
hypotheses. Early History Johannes Dzierzon,
a Silesian priest, in 1845 proposed the theory that drone bees (males)
developed from unfertilized eggs while workers and queens (both females) came
from fertilized eggs. The theory is based on facts that unmated and old
queens produce drone broods and that race-crossing produces drones like the
maternal race, while the daughters are hybrid. Dzierzon's Law was
strongly contested requiring him to defend his position through publication
(Dzierzon 1845, 1854). Dzierzon was aware of Mendel's
laws twelve years before Mendel published his work on peas. In 1854 he stated
that the drones of the second generation from a cross resemble either the
paternal or the maternal race, and that these two types occur in equal
numbers. He thereby visualized the fundamental gametic ratio (Dzierzon 1954). Dzierzon's law has been well
established as a rule for the honeybee with few exceptions. One of these is
the Cape honeybee of southern Africa, Apis mellifera var. kaffra.
This race produces females, both workers and queens, from unfertilized eggs
laid by workers (Jack 1916). The law applies to other insects of the order
Hymenoptera, including Vespidae, Formicidae, Ichneumonidae, Chalcididae and
Chalastogastra. Exceptions include unisexual species (males being unknown)
where the females reproduce indefinitely by parthenogenesis. There are also
some species which show alternation of unisexual and bisexual generations,
uniparental males and females occurring at one season, biparental females at
another. Sex Determination Cytology.--There is no evidence that males are developed from
fertilized eggs in any wild species of Hymenoptera. However, in the
honeybee, which is a domestic species, there are reports of biparental
drones; and laboratory cultures of Bracon hebetor Say indicate
the existence of biparental males. Females, on the other hand, are
usually produced from fertilized eggs, but as was previously mentioned may
come from unfertilized eggs. However, they always have the diploid number of
chromosomes. In general males develop from unfertilized
eggs and are azygotic. An azygote is an organism
that develops parthenogenetically from a haploid (reduced) nucleus. Studies
have revealed that in such azygotes originating from haploid cells, later
cleavages may result in doubling of chromosome number so that the adult would
be diploid and necessarily completely homozygous. For example, the chromosome
number of the male honeybee is characteristically 16 (Nachtsheim 1913). But
this is though to be double the haploid set since eight tetrads are found in
the first oocyte. The male may then be a diploid
azygote, with some male tissues having a even higher number of
chromosomes. Genetics.--Originally the principles of sex determination in
arrhenotokous species were though to be similar to Drosophila, where: Males = X; Females = XX In the honeybee, however, the
ratio of X-chromosomes to autosomes (not sex chromosomes) remains the same in
both sexes. In Drosophila the rates are different favoring a greater
amount of X-chromosome material in females, and males have more autosomal
material. In the principle of genic balance, it is thought that certain genes
tend to cause development in one general direction while other genes
counteract this trend. A character develops according to the resultant of
these genetic influences. However, since each gene is represented several
times in each cell and many times in the developing organism as a whole, the
only constant relationship must be on a ratio basis rather than on the basis
of an algebraic sum. Therefore, with sex determination in the honeybee, the
theory that the female has merely the equivalent or double the male set of
chromosomes (or genes) is not in agreement with the principles held for other
forms. Early Hypotheses
of Sex Determination.--Petrunkewitsch (1901) concluded after embryological
study that while the body of the male bee is haploid, the gonads are diploid
and derived from a fusion of two polar nuclei after maturation of the egg.
This was later disproved by Nachtsheim (1913). In the male honeybee (drone)
the first meiotic division does not involve the nucleus. There is merely a
small cytoplasmic bud of polar body given off.
The second division appears to be equal as regards the nucleus, but
practically all the cytoplasm remains at the one pole. The smaller cell or second
polar body degenerates and only one sperm cell is formed from a
spermatocyte. Castle (1903) first applied the Mendelian principle of
segregation to sex determination in the honeybee. He postulated differential
maturation not only for the egg but also for what he supposed, following
Petrunkewitsch, to be a reductional division of a diploid spermatocyte. A
pair of allelomorphic factors, maleness and femaleness, are concerned, with femaleness being
dominant. The female is heterozygous, but femaleness always passes into the
polar body, so that the unfertilized egg develops into a haploid male. The
testes, which are supposed to originate from a polar fusion nucleus, are
diploid and heterozygous for sex. Castle proposed maleness to pass into the
polar body in the maturation of the sperm, while dominant femaleness remains
in the sperm so that all fertilized eggs develop into females.
Nachtsheim (1913) suggested
that ancestral Hymenoptera may have been digametic in the male; but that when
parthenogenesis and male haploidy were acquired, the first spermatocyte
division became abortive so that no male-producing spermatozoa were
developed. Nachtsheim showed that the second spermatocyte division is
equational with respect to the chromosomes, as it is in the ants and wasps in
which the cytoplasm, unlike that of the bee, divides equally. He concluded
that the haploid set of chromosomes determines maleness, the diploid set
femaleness. He failed to find any constant difference indicating X and Y, and
suggested differential maturation of the egg directed by the presence or
absence of the sperm nucleus. This is comparable to Castle's idea except that
it is free of Petrunkewitsch's errors regarding the origin and composition of
the male gonad. Both Nachtsheim and Castle were
close to modern ideas of genic balance. Nachtsheim's final views that
the chromosome composition of the female is merely double that of the male, is
less accurate. Modern Hypotheses of Sex Determination.--Contemporary models that tend to explain sex
determination in Hymenoptera are (1) the single-locus, multiple allele model
(Whiting 1939), (2) multiple-locus, multiple allele model
(Crozier 1971) and (3) a genetic balance model (da Cuhna & Kerr 1957). Events leading to their
development are as follows: Bracon
hebetor [(Habrobracon
juglandis (Ashmead)] produces normal males from unfertilized eggs and
normal diploid females from fertilized eggs. Occasionally a normal diploid
female is produced by a virgin mother from crosses of certain stocks having
tetraploid oogonia (K. Speicher 1934). A gynandromorph may be produced
from a binucleate egg if one of the nuclei is fertilized. Male parts of the
body are, therefore, matroclinous, female parts biparental. Gynandromorphs
are also produced from uninucleate eggs in Habrolepis. If the parents are closely related,
diploid biparental males occur in relatively small numbers, the ratios
differing according to the stocks crossed (Bostian 1934). These diploid males
show no evidence of feminization either in external nor internal structures. Occasionally a haploid mosaic male
develops from an unfertilized egg laid by a female that is heterozygous for
one or more genes. These mosaic males show in different parts of the body the
alternative traits for which the mother was heterozygous (A. R. Whiting 1934).
A high proportion of the mosaic males show feminized structures in the
genitalia and more rarely in other parts (Whiting et al. 1934). On the basis
of eye color it was hypothesized that these feminized mosaic males are mosaic
for at least two sex factors. One type of tissue contains F.g.
(in the X chromosome) and the other contains allelomorphs f.G.
(in the Y chromosome). Either recessive factor causes maleness, but G.
produces some diffusible substance which, coming in contact with tissue
containing F., interacts so that feminization results (Whiting
1933a, 1933b). Two kinds of males were
postulated, F.g. (or X) and f.G (or
Y) which are phenotypically similar. The female contains both the X and the Y
chromosomes and is, therefore, heterozygous or digametic (F.g.
/ f.G.) or (X/Y). The dominant factors present in the two
types of males are complementary to each other in producing femaleness. Males
normally have one set of autosomes (1A) while females have two sets (2A). A female produces from
unfertilized eggs 1X + 1A and 1Y + 1A males in equal numbers. If crossed with
a 1Y + 1A male, she might be expected to produce from fertilized eggs females
1X + 1Y + 2A and diploid males 2Y + 2A, in equal numbers. Or, if crossed with
a 1X +1A male, the diploid sons should be 2X + 2A. These formulae show that
the genic ratio of X to A or of Y to A is the same in the diploid males as in
the corresponding haploid, while the female in unlike either, being a
combination of the two. Females are necessarily diploid, for they must have
both dominant factors F. and G. which are carried
in separate but homologous chromosomes. In 1943 Whiting elaborated on the
above and proposed a final scheme that was worked out by means of sex-linked
mutant genes as follows: Sex determination was shown to
depend upon a series of multiple alleles, of with 9 have thus farm
been identified (Whiting 1943). These are designated as xa, xb, ... xi. Any heterozygote (diploid),
xa/xb, xa/xc, xc/xd, etc., etc., is female. Any azygote (haploid) xa,
xb, xc, etc., etc., or homozygote
(diploid), xa/xa, xb/xb, etc., etc., is male. Normal females are heterozygous
for any two alleles of a certain series, while haploid males have any single
allele, and diploid males are homozygous for any one. The almost complete
sterility of the diploid males was found to be due to failure of the larger
diploid sperm to get into the eggs (MacBride 1946). Rarely occurring triploid
daughters of diploid males were also almost completely sterile. Manning (1949) suggested that
femaleness in the honeybee is a produce of a balance between a diploid
autosome set of 30 chromosomes plus an X chromosome, whereas maleness is an
effect of a haploid autosome set of 15 chromosomes plus an X chromosome. In
the formation of a sperm, the X chromosome is discarded so that each sperm
has only a set of 15 autosomes. Schmieder & Whiting (1947)
working with Melittobia, a close-crossed
chalcidid, suggested that in haplo-diploid species multiple
sex allelism may be the more primitive and general method
reproductive economy and that the close-crossed species have adapted some
other method. Melittobia is an exception which may fit an
"erroneous" scheme proposed by Lenhossek (1903) and Godlenski
(1910) for the honeybee. According to this scheme, the female produces two
types of eggs, of which only one type, the female-producing, is capable of
and requires fertilization; while the other produces males parthenogenetically. Da Cunha & Kerr (1957) put
forth the hypothesis of a series of male-determining genes in balance
with a series of female-determining genes. The female-determining (FD) genes
would be additive in their effect, whereas the male-determining genes (MD)
would not. Sex would be determined by the relation: 2FD > MD > FD The series of sex alleles of Bracon
hebetor studied by Whiting (1943) was interpreted as consisting of
female genes which have lost the property of determining femaleness unless
heterozygous (complementary multiple alleles). Evidence for this is
the fact that Bracon triploids are females (Torvik-Greb 1935, Inaba
1939). This hypothesis does not oppose the multiple allele one, but is merely
more general. Multiple alleles of Whiting (1943) are interpreted as
femaleness genes which lost the additive property. Laidlaw & Tucker (1964) came
out with the suggestion that female tissue in the honeybee was derived from
the union of two sperm only. Whiting (1967) studying the
pteromalid, Nasonia vitripennis (Walker), admitted that this
species did not fit her Whiting scheme.
Diploid males of Nasonia coming only from unfertilized eggs are
fertile and their triploid daughters are more so than the Bracon triploids.
The smaller number of chromosomes in Nasonia (n = 5; Bracon =
10) would provide a better chance for eggs of triploids to get the correct
representatives and correct number of chromosomes. That probability was
thought to explain their greater fertility. It may also involve the
production of smaller diploid sperms than those produced by diploid Bracon
males. Larger micropylar openings could also explain the fertility of diploid
Nasonia males. Finally, Crozier (1971) attempted
to integrate all mechanisms. In the summary of his paper, Crozier stated that
sex determination in haplo-diploid animals was explained by Whiting's scheme
for two cases only, and that the daCunha and Kerr genic-balance scheme, a
more general hypothesis, tended to explain sex determination for other
species. Crozier proposed a general hypothesis based on Snell's (1935)
multiple factor suggestion. This multiple-locus hypothesis
suggests that in haplo-diploid species, sex is determined by a number of
loci. Females are heterozygous at one or more loci, while males are
homozygous or hemizygous at all sex loci. At the molecular level, this effect
might be due to female-determining properties of heteropolymers formed
between the products of different alleles at any sex locus. Homopolymers or
heteropolymers between products at different loci are not formed or lack sex
determining activity. Haploid intersexes could arise from mutants that form
active homopolymers or active heteropolymers with products of other loci.
Diploid intersexes should be extremely rare, except in single locus species,
in which intersexes could result from mutations that reduce heteropolymer
formation. The data from a number of examples
support the multiple-locus hypothesis for Hymenoptera and haplo-diploid
Acarina, but not for coccids. No suitable data exist for other haplo-diploid
groups. Compared with single locus species, those with many sex loci will
have weaker selection operating on the alleles at each locus and will lose
fewer diploids as low viability males. Crozier concluded that testable
predictions for species with many sex loci indicate that prolonged close
inbreeding should yield diploid males; that diploid intersexes in outbred
lines should be extremely rare compared with haploid intersexes; and that
feminized borders, due to complementation between different sex alleles,
should often occur between genetically different blocks of tissue in gynoid
males. Luck et al. (1996) stated that the
single-locus and multiple-locus models both predict that diploid males will
appear when hymenopteran populations are continuously inbred. The genetic
balance model does not. In the single-locus model diploid males will occur in
one or two generations of inbreeding whereas several to many generations of
continuous inbreeding are required before diploid males will appear if the
multiple-locus model applies. Crozier (1971) argued that the absence of
diploid males following inbreeding cannot be taken as evidence that the
multiple-locus model is inapplicable because homozygosity at some sex
determining loci may be lethal. Experiments have documented that
the gender of Bracon hebetor Say is controlled by a single
locus (Whiting 1943), with nine alleles (Whiting 1961). Also the gender of
the honey bee, Apis mellifera L. (Woyke 1963), some Melipona
spp (Kerr 1974) and a sawfly, Neodiprion nigroscotum Midd.
(Smith & Wallace 1971) are all determined by a single locus with several
alleles. No cases are known in which multiple loci (multiple alleles) determine
the gender (Luck et al. 1992). Some Generalities
in Arrhenotokous Reproduction Biparental Males.--they are always
much less frequent then females, and are totally lacking when parents are unrelated.
When parents are related they may occur at a frequency of less than one
percent. However, in certain rare cases they may range to 25 percent (Bostian
1934). Biparental males never equal the
females as expected on a Mendelian basis, which is thought to be due
partially to a higher mortality among diploid males (Hase 1922, Whiting
1935). Their scarcity is largely explained by differential maturation of egg
nuclei. For example, if a Y sperm enters the egg, an X egg nucleus remains to
unite with it, other egg nuclei disintegrating and vice versa. King (1968)
gave evidence for the existence of accessory nuclei in certain hymenopteran
oocytes. Androgenesis.--was shown in Nasonia vitripennis by
Friedler & Ray (1951). Androgenesis is only artificially known, where
radiation inactivates the egg nucleus and the sperm nucleus dominates. In
this way a female can produce male offspring with paternate characters. Polyploidy.--has been demonstrated in Nasonia vitripennis
by Whiting (1959, 1960a). Generally, fertilized eggs develop into females and
unfertilized eggs into males regardless of the ploidy. The R locus.--in Nasonia vitripennis there is a short
region on one of the five chromosomes within which there are several factors
band between which no recombination occurs. Linkage is, therefore, complete
(Whiting 1956). Incompatibility Factors.--there are different cross incompatibility factors and
differing amounts of the same factor (Saul 1961, Whiting 1967). Sex Intergrades.--Two kinds occur (1) gynandromorphs and intersexes.
Gynandromorphs are often considered as genotypic mosaics in space. The
body regions differ genetically from one another and they are mostly
asymmetrical. Intersexes have been called phenotypic mosaics in time.
They start out development as one sex but change later on to the other sex or
to the possession of parts of the other sex. Intersexes are symmetrical. Other terms used in connection
with research on arrhenotoky are heterogony, which is cyclic
parthenogenesis; spanandry, in which males are absent or very rare,
and endomitosis where a doubling of the chromosome number occurs in
oogonial mitosis. Functional Aspects
of Arrhenotokous Reproduction In the biparental reproduction of
females and the uniparental production of males, Dobzhansky (1941) pointed
out that (a) there may be freedom to form gene combinations although the
supply of hereditary variations is limited, and (b) that functional haploid
males provide a means for the rapid elimination of unfavorable mutant genes
if the genes that are recessive in females have similar phenotypic effects in
both sexes. In contrast, where thelytokous
reproduction is solely involved, a phylogenetic blind alley may be
produced. Peacock (1925) pointed out that in the sawflies, a group in which
uniparental reproduction is of long standing, there is a stereotype of form.
Flanders (1945) showed how arrhenotoky may arise at irregular intervals in
the population of thelytokous-reproducing insects. Kelly and Urbahns (Webster
& Phillips 1912) showed evidence with Lysiphlebus testaceipes
where a switch to uniparentalism was produced. There is no direct
field evidence for the other way except Flanders (1965) produced an arrhenotokous
laboratory population in the thelytokous encyrtid Pauridia peregrina
Timberlake, and Stouthamer et al.( 1990) were able to "cure"
thelytokous populations of their thelytoky, thereby causing a reversion to
arrhenotoky. Rössler & DeBach (1972) give
convincing evidence to show that so-called thelytokous populations may not be
evolutionary blind alleys in that arrhenotokous reproduction is assumed
during certain intervals. This is probably the most detailed study performed
on a thelytokous population of parasitic Hymenoptera. Extranuclear Inheritance
and Polygenes in Arrhenotoky Inheritance of quantitative
behavior associated with gregarious oviposition (>one individual developed
per host) and fecundity in the South American parasitoid Muscidifurax raptorellus
Kogan & Legner (Kogan & Legner 1970) is accompanied by some unique extranuclear influences
which cause changes in the oviposition phenotype of females (Legner 1987a , 1987b; 1988a). Males are
able to change a female's oviposition phenotype upon mating, by transferring
an unknown substance (Legner 1987a , 1988a, 1988b). Females with the solitary genotype express gregarious
oviposition behavior after mating with males possessing the gregarious
genotype, and females with the gregarious genotype reduce the magnitude of
their gregarious behavior after mating with males of the solitary genotype.
The intensity of this response is different depending on the respective
genetic composition of the mating pair (Legner 1989a). Thus, the genes involved, by regulating phenotypic
changes in the mated female and aggression in her larval offspring, cause
partial expression of the traits they govern shortly after insemination and
before being inherited by resulting adult progeny (Legner 1987a , 1988a, 1989a). Such genes have been called wary genes and the
process by which they are inherited accretive
inheritance (Legner 1989a). Maternal inheritance of
extranuclear substances as discussed by Legner (1987a ) and Corbet (1985) does not explain the passage of traits
to offspring. Observations of linear additivity of the traits and variance
changes in hybrid versus parental generations and relatively constant daily
expressions of behavior in F1 and backcrossed populations, point
to chromosomal inheritance (Legner 1988a, 1989a,c). In the process of hybridization,
wary genes may serve to quicken the pace of evolution by allowing natural
selection for nonlethal undesirable and desirable characteristics to begin to
act in the parental generation. Wary genes detrimental to the hybrid
population might thus be more prone to elimination and beneficial ones may be
expressed in the mother before the appearance of her active progeny. If wary
genes occur more generally in Hymenoptera, their presence might account
partially for the rapid evolution thought to occur in certain groups of
Hymenoptera (Hartl 1972, Gordh 1975, 1979, 743-748), and possibly the quick
adaptation and spread of Africanized honey bees in South America as discussed
by Taylor (1985). As discussed earlier, the ability
to change the adult female's expression of a quantitative character, either
positively or negatively, challenges accepted views of polygenic loci, and it
may be that such loci are not in fact inherited, but rather another group of
genes which have the capability to switch on or off the loci.
Such genes may influence DNA methylation of the loci controlling oviposition
behavior, as shown for other organisms (). All polygenic loci may be
perpetually present for a given quantitative trait in all individuals of both
Muscidifurax raptorellus races, but they are either activated
or inactivated by substances under the control of another group of genes. Further studies in 1995 by
Stouthamer et al. (unpublished) have shown the involvement of larval
cannibalism and much greater complexities in this species' reproduction. An
account may be found in <aggress.htm> Recombinant Hymenopteran
Males Some unique considerations are
required in the formation of recombinant males of haplo-diploid breeding systems.
Although normal oogenesis in arrhenotokous Hymenoptera does not deviate from
that found in diploid-diploid organisms, hymenopteran spermatogenesis is
highly modified (Crozier 1975). Because hymenopteran males are haploid,
marked modifications of spermatogenesis are necessary to ensure that a
balanced set of chromosomes is transmitted via the sperm. The principal
difference is that the first division is somewhat abortive, with no
karyokinesis, so that there is only one equational division (Crozier 1975).
In most Hymenoptera, the sperm of any one haploid male are identical, at
least in the genetic components they carry. Considering a hymenopteran example
involving only two loci in which parental cohorts are homozygous for
different alleles at each locus, the F1 generation of females
would be genetically identical and heterozygous. Assuming that the loci in
question are unlinked, each F1 female would be capable of
producing four kinds of gametes: AB, A'B, AB' and A'B',
in equal proportions. Similarly, such virgin F1 hymenopteran
females produce four haploid and genetically distinct males from unfertilized
eggs: AB, A'B, AB' and A'B'. However, 50% of
these males would be of the parental genotypes (eg., AB & A'B'), as
opposed to none of the F1 females. In this way the recombinant
hymenopteran males differ from diploid-diploid systems: there are different
kinds of genotypes depending on the number of active loci. When crossing F1
females with males produced by that generation (a practice necessary in
estimating the number of active polygenic loci) each free-living, haploid
recombinant male produces only a single type of gamete, but among the population
of males present, all gametes that are produced by the F1 hybrid
female also will be represented. However, at this point each of the different
kinds of males (four in the above example) must have equal mating advantage,
which must be guaranteed by manual random selection. Also, where large
numbers of genetic loci are involved, it is essential to have a sufficient
number of replicates to ensure that the larger number of male genotypes are
given equal statistical chance in mating. Estimations of
the Number of Active Polygenic
Loci The minimum number of independent
genes with additive effects that contribute to the expression of a
quantitative trait, such as cannibalism intensity, can be estimated from the
means and the variances of the character in the parental cohorts, their F1
and F2 offspring, and backcrossing data, by applying Wright's
(Castle, 1921) formula: nE = (up2
- up1)2 / (8o2s) < n [ nE
= effective number of genetic factors up1 =
mean of parental cohort-1 up2 =
mean of parental cohort-2 o2s
= difference in variances between compared generations (see Lande 1981) Four estimates and their standard errors are derived from
Lande's (1981) method as follows: nE1 considers F1 and
F2 variances; nE2, the F1, F2 and
P2 variances; nE3, the F2 and first and
second backcross variances; and nE4, the F1, P1,
P2 and first and second backcross variances Assumptions necessary for the
accurate application of Wright's method enumerated by Lande (1981) and Wright
(1952) are that the two parental populations have homologous gene sequences
so that there is no post-mating reproductive isolation due to chromosomal
rearrangements; any number and frequencies of alleles are allowed at each
locus within the parental populations; and the loci or segregating factors
are not linked and in random combination in each parental population, with no
significant selection during the experiment. Also, all mating individuals
must be chosen at random from the respective populations, and there is
semi-dominance at all loci, which all make equal contributions (Wright 1968). Analysis Scale.--the
scales for analysis should guarantee additivity of the mean phenotypes in F1,
F2 and backcross populations, and there should be a linearity of
P1, F1 and P2 variances when plotted against their
means, with the extra variance segregating in backcross populations being
about half that in the F2 (Lande 1981). The best scale for analysis is one
on which the effects of both genetic and environmental factors are as nearly
additive as possible, although because of a complex of genetic and environmental
factors, these effects are in general not additive (Wright 1968). However,
whenever interaction effects exist, there is no single transformation that
satisfies all available criteria of additivity. Transformations for the data may be
selected with the procedure outlined in Wright (1968) as follows: Standard
deviations are regressed in terms of means among inbred, presumably isogenic,
parental cohorts and their F1's in order to derive a regression
formula Y = a + bx. Then the relationship a/b suggests the
transformation function. Coefficient of
Heritability Two methods may be employed to
estimate the coefficient of heritability, which is the ratio of the additive
genetic variance to the phenotypic variance. The first method considers
heritability in the broad sense (H), and assumes that inbred parents
and the F1 are genetically homogeneous, so that all variance
observed therein is due to environmental influence. An overall value for environmental
variance is derived by averaging the variances for one female and the F1.
This value subtracted from the total variances, represented by the F2
variance, gives an estimate of genetic variance. Then genetic variance
divided by total variance estimates heritability (Goodenough 1984). Standard
errors of H may be calculated with Tukey's Jackknife
method, explained in Sokal & Rohlf (1981). These estimates
measure the extent to which individual differences in the population are due
to differences in genotype. They represent all the genotypic variance
including the additive, dominance and epistatic kind. Estimates may also be made of
heritability in the narrow sense (h2) by regressing
expressions of behavior of female offspring on one of the female parents
(Falconer 1981, Owen 1989). The covariance is then computed from the
cross-products of the paired values. Covariance is then divided by the
variance among the parental females and this value is doubled for an estimate
of h2 (see Owen 1989 and Hellmich, et al. for hymenopteran
breeding systems). Because dominance can influence
estimates of gene number by distorting the expression of the phenotype, the
various hybrid and backcross cohorts must be examined for its presence. The
dominance level (D) in F1 progeny may be estimated using the index of Stone
(1968), which was derived for single loci, but has been used in polygenic
systems (Raymond et al. 1986). the P <0.05 confidence limits can be
derived from formulae in Misra (1968). The parameter "D" may vary
linearly from +1, indicating complete dominance, to -1 indicating complete
recessivity, and 0 indicating perfect codominance. Stone's (1968) formula: D = (2
log F1 - log P1 - log P2 / (log P1
- log P2) Some Generalities
in Thelytokous Reproduction Thelytoky is not common among
animals, and White (1984) estimated that only 1,500 records are known.
Thelytoky was reviewed for Hymenoptera by Phillips (1903), Winckler (1920),
Vandel (1928), Clausen (1942), Slobodchikoff & Daly (1971) and Crozier
(1975), where about 100 cases are known. Recently Stouthamer (1990) showed
that at least 270 reported cases exist in Hymenoptera, not including the
2,000 cases of cyclic thelytoky found in Cynipoidea (Herbert 1987). Luck et al. (1996) stated that thelytoky is much more
prevalent than generally thought. The family Aphelinidae shows a large
percentage of the species with thelytokous populations. DeBach (1969)
observed that the genus Aphytis had 30% of its species demonstrating
this mode of reproduction and the family Signiforidae showed 40%. Causes of thelytoky are not always
generally well understood. Two possible genetic mechanisms may lead to
thelytoky. Thelytoky as a simple mendelian or polygenic trait, or thelytoky
resulting from epistatic interactions between genes (Luck et al. 1996).
Little information exists on the genetic causes of thelytoky, hybridization
leading to thelytoky may be caused by epistatic interactions between genes.
Thelytoky as a simple recessive mendelian gene has been indicated to occur in
the Cape honey bee Aphis mellifera carpensis Ersholtz,
although Kerr (1962) reported that thelytoky in that species is not that
simple. Hybridization leading to thelytoky
has been reported twice in Trichogramma. Nagarkatti (1970) crossed a
female of Trichogramma perkinsi Girault with T. californicum
Nagaraja & Nagarkatti male. This cross produced 17 offspring in the F1
generation. One of the females was thelytokous and the other seven females
were arrhenotokous. A similar example was reported by Pintureau & Babault
(1981). In crosses between T. evanescens Westwood and T.
maidis Pintureau & Voegelé the F1 hybrid females reproduced
by thelytoky. Their F2 offspring reproduced by arrhenotoky,
however. Hybrid induced thelytoky has also been reported in Muscidifurax
raptor Girault & Sanders (Legner 1987a ,1987b). Hybridization increased levels of tychoparthenogenesis
(occasional production of female offspring from unfertilized eggs) in Bracon
hebetor (Ashmead) (Speicher 1934). Luck et al. (1996) refer to an
unusual case of thelytoky induction in the Aphidius colemani
complex (Tardieux & Rabelasse 1988). Thelytoky was induced in certain
cases when males attempted matings with females from different geographic
locations. Electrophoretic observations with females that were not
inseminated by these males showed that the female offspring of the
"cross" had indeed the maternal genotype. Typically, the genus Muscidifurax,
attacking synanthropic Diptera, also shows completely parthenogenetic modes
of reproduction in some geographically isolated populations. In Muscidifurax
thelytoky is automictic which includes meiosis and the process of
endomitosis, or endopolyploidy, where chromosomes are duplicated without
division of the nucleus, resulting in increased chromosome number within a
cell. Chromosome strands separate but the cell does not divide. Endomitosis
in M. uniraptor Kogan & Legner has been observed to occur
as late as the 2nd cleavage stage in eggs that were already deposited in the
host (Legner 1987a ,1987b). In the studies on Aphytis mytilaspidis
by Rössler & DeBach (1972a,b; 1973), it was shown that thelytokous forms
of Hymenoptera are not completely reproductive isolated from sibling
arrhenotokous forms. The greatest barrier to interbreeding seemed to be the
precopulation period, where arrhenotokous males spent a greater length of
time in courtship with thelytokous females. There was a tendency for the
thelytokous form to be replaced entirely by arrhenotokous forms in the long
run; and persistence of thelytoky seemed dependent on the hybrids finding
suitable environmental conditions, such as host type. in Muscidifurax,
thelytoky may be transferred to an arrhenotokous population in two ways: (1)
by mating adventitious males from a thelytokous population to virgin hybrid
females of an arrhenotokous population and (2) by backcrossing a hybrid
female of interhemispheric origins to males of one of the original parents
(Legner 1987a ,1987b). The first method is apt to be more successful than the
second one. However, the second method fits the pattern most often ascribed
to the origin of thelytoky in animals: hybridization between two related
bisexual species. The question of whether only
chromosomal inheritance is involved in the acquisition of thelytoky in
Hymenoptera is uncertain, and there is mounting evidence to suggest that the
process may also include extrachromosomal phenomena (Legner 1987a ,1987b; Stouthamer 1989, Stouthamer et al. 1990, 1993). Although
adventitious males from thelytokous populations may simply transmit a
dominant nuclear gene for thelytoky, there is also the possibility that
thelytoky could involve infection by microorganisms found in the reproductive
tract. Such organisms or their products would be capable of initiating the
endomitotic process, resulting in parthenogenetic female offspring. There is an apparent relationship
to the titre of the causative factor in thelytoky. For example, production of
thelytokous females in M. uniraptor is greatest when
oviposition is interrupted for 24 hours by scheduling host presentation on
alternate days or by slowing oviposition rates during early adult life. Such
interferences allow the titre of the factor to rise. Higher concentrations of
microorganisms may thus guarantee a greater proportion of thelytokous female
offspring. It could reasonably be assumed that microorganisms and certain
chemicals produced by them are involved, with the latter inducing
endomitosis. Heat treatment (32B
C for >24 hr) beginning at a critical stage in oocyte formation, blocks
endomitosis and male progeny result. If any enzymes, microorganisms or both
were involved directly or indirectly in promoting endomitosis, the prolonged
exposure to higher temperatures could kill or inactivate them. Some work
points to their probable residence in or near oocytes which are in later
developmental stages. Such observations tend to preclude
a wholly genetic aspect to thelytoky. If, for example, microorganisms and
accompanying chemicals, or inducing enzymes which they produce, are
transferred to the developing ova, endomitosis might be influenced in the
next generation, and thelytoky would be passed on without genetic change.
With such a system it is possible to envision quantitative variation in
microorganisms and enzymes and hence the number of thelytokous females
produced. Because the titre appears to build up during host-free periods,
microorganismal multiplication and/or elaboration of the chemical substances
would have to proceed relatively slowly. The possibility might be considered
that in the presence of a gene for thelytoky, microorganisms may play a role
in directing cytological processes towards a production of parthenogenetic
females. Microorganisms involved in the production of thelytoky have been identified molecularly by Stouthamer et al. (1993). They comment that inherited microorganisms are widespread in insects, having been implicated as causes of female parthenogenesis and cytoplasmic incompatibility. Normal sexual reproduction can be restored by treatment with antibiotics. Sequence analysis of the DNA encoding 16S ribosomal RNA show that cytoplasmic incompatibility bacteria from diverse insect taxa are closely related, sharing 95% sequence similarity. They belong to the alpha subdivision of Proteobacteria. Stouthamer et al. (1993) show that parthenogenesis-associated bacteria from parasitoid Hymenoptera fall into this bacterial group, having up to 99% sequence similarity to some incompatibility microorganisms. Both incompatibility and parthenogenesis microorganisms alter host chromosome behavior during early mitotic division in the egg. Incompatibility bacteria act by interfering with paternal chromo |