Our discussion of insect reproduction seemed to assume female insects were sort of automatically endowed with sperm at the correct times in their lives. This is not always a correct assumption. Indeed, a certain proportion of adults of most insect species fail to locate mates. The business of locating mates, courtship interactions, mating, fertilization, depositing eggs, and caring for and provisioning offspring (or not doing these activities) are aspects of mating systems. Many of these components of mating systems are mediated through neuroendocrinological mechanisms. The idea of neuroendocrine systems draws attention to the physiological basis of insect mating systems. A very common example comes from considering the roles and actions of sex pheromones in the mate-seeking phases of mating systems.

Lurking behind these components, or phases, of mating systems is still another aspect of insect reproduction. Males have another evolutionary issue. Beyond locating, courting, and mating females, males are constrained by a need to assure the paternity of self. Strong selective pressures seem to favor mechanisms that assure an individual's paternity. I would just speculate that individuals lacking paternity assurance mechanisms would probably not be well represented in succeeding generations. Insects express many behavioral mechanisms, such as mate guarding, to assure an individual's paternity. Insect also elaborate a number of biochemical and physiological mechanisms to enhance an individual's chances of assuring paternity. Males and females seem to cooperate in some of these mechanisms.

This lecture is designed to give a sense of the close connection between physiology and behavior in insect systems. We will consider a mating system because I have a fair bit of experience in this work, and because there sufficient information to present something close to a detailed picture of the system. The mating system of the Austrialian field cricket, Teleogryllus commodus, features behavioral and biochemical mechanisms of assuring paternity.

Nearly everybody knows that male crickets attract their potential mates by emitting a calling song. You may recall the case of an American president's wife asking the Secret Service to remove a singing male from her bedroom in the White House. Singing is an expensive business for male crickets. Males often maintain and defend a territory that, I suppose with luck, will later serve as a courtship and mating arena. Male crickets produce their songs by stridulation of the their wings. The singing itself is an energy-intensive process. Before they begin singing, males of some crickets create a small burrow. The burrow acts as a sort of amphitheater, extending the males' broadcast distance. I have often wondered what it is that females hear. Does the song of a conspecific male sound like some kind of recording star? Maybe a Bob Dylan or a Pavarotti?

We begin with the calling song because the temporal organization of male and female mating behaviors increases the chances of successful mate location for both sexes. First, calling behavior is under circadian rhythm control. Males express their calling behavior on a daily basis in a predictable rhythm. Of course, females are attracted to the calling song. Males must be prepared to court and mate with a female by the time he begins his singing. There are physiological and behavioral preparations. Again, many males prepare a burrow. There is more. As you already know, most (but not all) cricket species mate by transferring a spermatophore from male to female during copulation. Males of T. commodus synthesize a new spermatophore each day. As seen for singing, spermatophore synthesis is also under circadian control. In the cricket I worked on, spermatophore synthesis takes place in late afternoon, before nightly singing. The point to take is that two aspects of the mating system are expressed in a regulated temporal organization.

Female crickets also exhibit a number of sexual behaviors. One is her nightly locomotor behavior. We can show this in an actogram. The top bar shows a 12:12 hour photoperiod. We take three points. First, virgin females express a nightly locomotor behavior, that is, they go out walking around the park. We might presume they walk around listening for calling songs. The temporal organization of the female walking happens to match up with the male singing rhythm. Hmmm..

The second point is shown in the column under 'S'. The call song can stimulate virgin females into locomotor activity is the song is played during normally inactive periods. I guess this helps out in cases where the clocks are set just right. The third point is that after mating, the female is no longer responsive to calling songs, and she no longer expresses here nightly locomotor activity. The main point here is the sexual berhavior of males and females is characterized by a temporal organization that facilitates mate finding.

As just mentioned, females change their sexual behaviors after mating. As mentioned, females are no longer stimulated to walk by calling songs, and the nightly locomotor behavior ceases. We might think these two points make sense because a mated female no longer needs to risk her life walking around in dangerous parks. These behavioral changes also facilitate paternity assurance because the a mated female is more likely to remain in the male's territory. Females also exhibit a third behavior right after mating. There is a very large increase in egg-laying behavior, usually in the first 24 hours following mating.

This is shown in a figure. We have a three-dimensional plot of egg-laying behavior. The x-axis indicates individual females. The y-axis indicates numbers of eggs deposited by each individual, and the z-axis shows four time points. The first x-axis represents egg-laying behavior in the 24-hour period before mating. This shows that sexually mature virgin females will typically deposit a small number of sterile eggs. The next axis represent the first 24 hours after mating, during which most individuals have deposited a vastly increased number of eggs. The next line shows some females will wait for another 24 hours before laying her eggs. I guess this occurs more rarely than suggested by these data, because the nearly all the females I have experienced will deposit their eggs during the first night.

The burst of egg-laying activity has been called an "egg-flood" because so many eggs are deposited. This is a misleading term because each egg is deposited in a controlled behavioral program. The eggs do not "flood" out of the females. The main points here relate to females. The temporal organization of female sexual behavior facilitates mate finding, and mating radically alters female behaviors.

These changes in female sexual behavior, reduced response to male calling songs, reduced locomoter activity and a sustained program of egg-laying, are cooperative. Together, these changes form the characteristic post-mating behavioral syndrome. The physiological basis of changing the first two behaviors remains unknown. On the other hand, the mechanism of releasing oviposition behaviors is better understood. We know that the release of egg-laying behavior is not physiologically coupled to the other behaviors.

Let us consider the anatomy of cricket female reproductive tracts. In this drawing one ovary is absent to show structures that otherwise would be obscured. OP indicates the ovipositor, GC the genital chamber, ACG the accessory glands, M the oviducal muscles, LOD the lateral oviduct, LAG the last abdominal ganglion, SPD the spermathecal duct, SP the spermatheca and OV the ovary. A successful mating entails placing a spermatophore into the genital chamber. A fine spermatophore tube makes its way through a pore in the ceiling of the genital chamber, and up into the spermatheca duct. This step is necessary for complete transfer of the seminal fluids into the spermatheca. A fair proportion of matings fail on this point, and examination of spermathecae from these failed matings shows the seminal fluids did not reach the spermatheca.

Aside from sperm, the enzymes and substrate required for biosynthesis of PGE2 are transferred along with the seminal fluids. Once the seminal fluids reach to spermatheca, the transferred enzyme activity converts arachidonic acid into PGE2. The PGE2 moves out from the spermatheca and into general circulation. I believe, with knowing for sure, the circulating PGE2 releases egg-laying behavior by interacting with specific receptors in the last abdominal ganglion. This is known as the enzyme transfer model of releasing egg-laying behavior. The model is pretty well supported, although we do not have data on the last point.

Here is a summary of the evidence on the enzyme transfer model.

- PGE2 releases egg-laying behavior in sexually mature virgin females

- Spermathecae from mated females contain about 500 ng PGE2/spermatheca. Those from virgins had no detectable PGE2

- Spermathecae from mated females synthesized PGE2 at substantial rates, over 30 pmol/hr; those from virgins synthesized none

- Spermatophore contents synthesized PGE2 at about the same rates seen in females, 25 pmol/hr. The spermatophores contained almost zero PGE2

On the first point, when PGE2 was injected into the hemocoel of sexually mature virgins, the females responded by releasing egg-laying behavior during the following 12 hours. Second, PGE2 could be detected in spermathecae from mated, but not virgin females. Spermatophores themselves contained only very small amounts of PGE2, indicating that PGE2, per se, is not transferred to females during mating. third, PGE2 biosynthetic activity was detected in spermathecae from mated females, and in spermatophores, but not in spermathecae from virgin females. Taken together, these data strongly supported the enzyme transfer model.

The next question to ask relates to the specificity of PGE2 in releasing egg-laying behavior. Do all PGs release the behavior, or is the action specific to E-type PGs? We understand from our consideration of receptor sites and signal transduction mechanisms this is an important question. If the PGs act in a receptor-mediated physiology, then we would expect to observe specificity to E-type PGs. Let us consider structures of PGs. All 1-series PGs are formed from 20:3n-6. The 2-series PGs are formed from 20:4n-6, and the 3-series PGs are made from 20:5n-3. PGs of each series have 2 fewer double bonds than their parental fatty acids. The key point is most PGs look pretty much alike. They are all 20-carbon carboxylic acids with substitutions on C-15 and a 5-membered ring. The substitutions on the ring define the letter series of each PG.

To investigate the relationship between PG structure and egg-laying activity, we injected a standard dose of PG into the hemocoel of sexually mature virgins, then observed the egg laying responses. We worked with 16 different compounds. We arranged these structures into three groups, based on structural features. The compounds in group 3 are not the standard PGs. We tested the egg-laying activities of a monounsaturated fatty acid, to control for a general pharmacological effect of lipids. We used a PG that is sterically indered by an oxygen bridge, and a lipoxygenase product that lacks the 5-membered ring of PGs. None of these compounds release egg-laying behaviors.

At the other extreme, highest egg-laying activity was associated with the group 1 PGs, all variations on PGE2. All feature a 5-membered ring, and in every case the ring is substituted with a C-9 keto and a C-11 hydroxyl. These particular ring features are important. We can compare the egg-laying activity of other PGs lacking this arrangement, those in group 2. The group 2 PGs lack the PGE features, and these yielded zero to only low egg-laying activity. Overall, there is a biological specificity for E-series PGs. Again, this finding is consistent with the idea that the PG action is mediated through receptor sites. The receptor sites can discriminate among closely related molecules.

The enzyme transfer model indicates the enzyme activity from males converts arachidonic acid into PGE2 within the spermathecae of newly mated females. The PGE2 subsequently moves into circulation, then releases the behavioral program of egg-laying. One of the most direct tests of this idea would be to place radioactive arachidonic acid into spermathecae of mated females, then see if radioactive PGs turn up in their hemolymph. The practical problem is it is next to impossible to inject anything into a spermatheca. After trying this a few times, I conceived the idea of letting a male cricket inject radioactive arachidonic acid into spermathecae.

I wanted to determine the radioactivity was associated with arachidonic acid, and not some uncharacterized metabolic by-product. I collected a bunch of spermatophores, then extracted total lipids from them. After isolating phospholipids on thin layer chromatography, we prepared methyl esters of the fatty acids. We analyzed these by radio-high performance liquid chromatography. There was a single peak of radioactivity corresponding exactly with authentic arachidonic acid. So the males somehow packaged radioactive arachidonic acid into the phospholipid fractions of their spermatophores.

This work set the stage for the experiment I wanted to perform. I was interested in seeing the actual sexual transfer of arachidonic acid from male to female. To look at this I allowed my pre-labeled males to court and mate with untreated virgin females. It usually takes about an hour for the contents of a spermatophore to ooze into a spermatheca. One hour after mating with a pre-labeled male, I isolated the spermatheca from each female. The spermathecae from these matings contained about as much radioactivity as a typical radioactive spermathophore. On the other hand, about 24 hours after mating with pre-labeled males, the spermathecae contained no radioactivity. We see the males can transfer radioactive arachidonic acid to females.

The overall data are consistent with transfer of radioactive arachidonic acid from male to female, followed by movement of the radioactivity from the spermatheca into the hemolymph. Of course, the next experiment was to collect hemolymph from females mated with pre-labeled males, and characterize the radioactivity in the hemolymph. Most of the radioactivity in female hemolymph was associated with PGE2 or polar products of PGE2 metabolism. These findings indicate the PGE2 in the hemolymph of mated females is derived from arachidonic acid from males.

Now, this work was done on a single cricket species, Teleogryllus commodus. Overall, the work supported the enzyme transfer model, now amended to indicate males transfers and enzyme and substrate for PG biosynthesis in females. We might think this sort of mechanism occurs in many insect species, but it is not so. PG levels are greatly increased in the female reproductive tracts of many insect species. These include:

house cricket

The mechanisms of increasing PG levels differ among species. As you might expect, the mechanism in the house cricket and the locust is just like the Australian field cricket mechanism. The moths transfer preformed PGs, per se, in their matings. Males of the housefly mobilize and transfer arachidonic acid, but not the enzyme, during their matings. In all cases, PGs increase in female reproductive tracts following mating.

Do these PG increases always release egg-laying? The answer is certainly not. This has been tested in quite a few species, and so far only crickets and the silk moth seem to positively release egg-laying in response to PGs. We conclude that PGs are involved in other, still unidentified, aspects of insect reproductive biology.

Let us return to our theme of assuring paternity, by asking what is the biological significance of the cricket mating system? First, the reproductive success of an individual male is related to how many of his sperm are successful in fertilizing eggs, not to how many females acquire his sperm. By casting the issue in terms of sperm success, rather than mating success, we introduce an area of evolutionary biology known as sperm competition. Mechanisms to avoid or reduce sperm competition are recognized as a powerful force in the evolutionary shaping of mating systems.

Two conditions must obtain to allow the occurrence of sperm competition. One, females must be able to mate with more than one male. Two, sperm must remain viable in the body of the female. Sperm competition is not at all restricted to insect biology. There are many examples of the influence of sperm competition in other invertebrates as well as vertebrate mating systems.

One of the basic experimental strategies in assessing sperm competition is known as multiple mating experiments. In these experiments a single female is allowed to sequentially mate with two or more males. Among insects, the general finding is that the first male does not always fare quite so well. In many experiments the last male's sperm fertilizes 75 to 100% of the eggs. Here are two examples of strategies to increase chances of the mating success of the last male. Fruit flies displace sperm from previous mating. Similarly, locusts "flush" sperm from previous mating. These actions serve to reduce the competition for egg fertilization posed by the sperm populations already in a female's possession. There are many adaptations, behavioral and physiological, that appear to give a competitive edge to one's own sperm, relative to another population of sperm. Some of these adaptations seem to work in concert. The cricket mating system includes a biochemical mechanism to release egg-laying behavior. For another component of the same system, males perform their calling, courtship and mating in a pre-constructed arena. Due to her own syndrome of post-mating behaviors, the females tend to remain in their arenas. For still another component of the system, males tend to guard their newly mated females until the eggs are deposited.

1. PLUGS

These serve as physically barriers, blocking the entry of additional sperm populations. These are seen in various insects, spiders, snakes and mammals.

2. PROLONGED COPULATION

The male himself acts as a sort of plug. This is seen in many insects and in certain vertebrates.

 

3. POST-MATING GUARDING

Seen in many insects, including dragonflies, dungflies and in the cricket system.

 

4. TAKE-OVER AVOIDANCE

Includes going to places of low risk, and maintaining territories.

 

5. TRANSFER CHEMICAL AGENTS ALTERING BEHAVIOR