FILE: <BC-16

Sex Determination in Animals

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.

The csd gene

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.

Multiple-locus CSD

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 / (8o²_s) < n

[ n_E = effective number of genetic factors

up₁ = mean of parental cohort-1

up₂ = mean of parental cohort-2

o²_s = difference in variances between compared generations

(see Lande 1981)

Four estimates and their standard errors are derived from Lande's (1981) method as follows: n_E1 considers F₁ and F₂ variances; n_E2, the F₁, F₂ and P₂ variances; n_E3, the F₂ and first and second backcross variances; and n_E4, 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).

Dominance

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 chromosome incorporation in fertilized eggs, while parthenogenesis bacteria prevent segregation of chromosomes in unfertilized eggs. These traits are adaptive for the microorganisms. Judging from their sequence similarities, Stouthamer et al. (1993) concluded that parthenogenesis bacteria and cytoplasmic incompatibility bacteria form a monophyletic group of microorganisms that specialize in manipulating chromosome behavior and insect reproduction.

Consequences of Thelytoky.--Luck et al. (1996) summarize the outcomes of thelytokous reproduction. They point out that (1) females do not have to mate to produce female offspring and (2) all the offspring of thelytokous females are female. The first difference gives thelytokous females an advantage whenever their arrhenotokous counterparts are not able to find males (Tomlinson 1966, Gerritsen 1980, Cornell 1988)., Assuming that thelytokous females are equally as fecund as their arrhenotokous counterparts, the second difference leads to a higher intrinsic rate of increase in thelytokous compared with arrhenotokous females (Timberlake & Clausen 1924, Vet & van Lenteren 1981).

In studies where the fecundity of thelytokous populations are compared with arrhenotokous populations, the results are not always as expected, however. Smith (1941) found that thelytokous females of the spruce sawfly Diprion polynotum Ht. produce about a third of the offspring produced by arrhenotokous females. There was some question here whether both were actually of the same species, however. In work by Rössler & DeBach (1972, 1973) on Aphytis mytilaspidis (LeBaron), males from an arrhenotokous population were mated with females from a thelytokous population, and the hybrid offspring formed differed in their host preference. The thelytokous strain preferred Latania scale, Hemiberlesia lataniae (Sign.) and the arrhenotokous strain preferred cactus scale, Diapis echinocacti (Bouche). The number of offspring produced by the thelytokous strain was less on cactus scale while on Latania scale it was more (15 / 22 vs 12/0.89, respectively). Stouthamer (1989) compared the offspring production of thelytokous lines and arrhenotokous lines that had been derived from them by antibiotic treatment of Trichogramma deion and T. pretiosum. The results indicated that in all cases the total offspring production of the arrhenotokous line was significantly higher than that of the thelytokous line. Legner & Gerling (1967) comparing host-fed and host-deprived Muscidifurax uniraptor Kogan & Legner cultures found that the host-deprived females had a higher fecundity.

Exercises:

Exercise 16.1--Discuss how arrhenotoky was first discovered.

Exercise 16.2--Be able to discuss in detail the various hypotheses proposed for sex determination in arrhenotokous arthropods.

Exercise 16.3--Describe the following: biparental males, androgenesis, polyploidy, sex intergrades.

Exercise 16.4--Compare the functional aspects of arrhenotoky with thelytoky.

Exercise 16.5--In a hypothetical case, Bracon hebetor is imported to America from Europe for the biological control of an invaded lepidopterous pest. In its native home B. hebetor is shown to be responsible for the extremely low density of the pest in question. However, in America only partial control was achieved. Basic studies revealed that (1) the sex ratio of B. hebetor in America averages only 20% females while in Europe it averages 80% females; and (2) experiment station reports show that the original culture of the parasitoid was obtained from a single mated female in Europe.

What might be wrong with the population of B. hebetor in America? What could be done to correct the situation and possibly attain a higher degree of biological control?

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Sex Determination (Animals)	Sex Intergrades
In Hymenoptera	Functional Aspects of Arrhenotokous Reproduction
Early History	Extranuclear Inheritance and Polygenes in Arrhenotoky
Cytology	Recombinant Hymenopteran Males
Genetics	Estimations of the Number of Active Polygenic Loci
Early Hypotheses	Coefficient of Heritability
Modern Hypotheses	Dominance
Generalities in Arrhenotokous Reproduction	Some Generalities in Thelytokous Reproduction
Biparental Males	Consequences of Thelytoky
Androgenesis	Exercises
Polyploidy	References
The R locus	[Please refer also to Selected Reviews & Detailed Research ]
Incompatibility Factors