BIO315 Laboratory Guide #8
REPRODUCTIVE BEHAVIOR IN
PARASITOID WASPS
The jewel wasp Nasonia (Mormoniella) vitripennis is a small, stingless solitary wasp which is a reproductive parasite of several species of large flies. In the wild, female jewel wasps seek out the nests of songbirds, and oviposit their eggs into the developing pupae of the flies whose larvae inhabit those nests. In the laboratory, jewel wasps will readily accept the pupae of the common blowfly Sarcophaga bullata as a substitute.
Each female wasp finds and inspects a suitable host fly pupa, drills through the puparium (pupal case), extends her ovipositor through the drill hole, and lays up to 25 eggs against the outer surface of the enclose fly pupa. Within 2 days the wasp eggs hatch into larvae which attach to the fly pupa and begin to consume it from the outside. The wasp larvae grow rapidly and metamorphose into pupae by the 9th day. At this point only a dry husk is left of the fly pupa. By the 12th to 14th day the wasp pupae eclose from their pupal cases as sexually mature adults and quickly break through the fly puparium.
Both male and female wasps have wings, but only the females are capable of flight. Males and females begin mating soon after eclosion, so that mating often occurs with siblings. Soon after mating females begin seeking a new host fly puparium in which to oviposit. Females may oviposit successfully in several hosts. Conversely, a single host fly puparium may be oviposited by more than one female.
Several additional features of jewel wasp reproductive biology make them particularly interesting subjects for tests of natural selection theory:
1) Jewel wasps, like all Hymenoptera, are haplodiploid. Females are diploid, develop from fertilized eggs, and receive half their genes from each parent. Eggs are produced by meiosis. Males, on the other hand, are haploid, develop from unfertilized eggs, and receive their genes entirely from their mother. Sperm are produced by haploid mitosis.
2) Mated females store sperm in an internal spermatheca and can control the numbers of fertilized and unfertilized eggs which they oviposit in each host fly puparium. Unmated females can produce only male offspring.
3) Females can determine whether or not they are the first wasp to oviposit in a given fly puparium. This means that a female wasp could potentially adjust the sex ratio of her offspring to maximize her reproductive fitness (most conveniently assessed as the potential number of her grandchildren), based on the likelihood that she will be sharing a given fly puparium with another female wasp.
4) There are pure-strain single-locus eye-color variants in jewel wasps which allow the experimenter to determine the parentage of offspring wasps.
Haplodiploid genetics is a bit of a mind-bender when applied to the Fischer-style calculation of heritability and relatedness. Try using the simulation Foundress to determine sex ratio strategies for first ovipositors and second ovipositors. Not to spoil the surprise, but you should discover the following basic rationales:
1) The best “strategy” for the first wasp ovipositing in a puparium (foundress #1), especially at low wasp population densities, would be to produce a “litter” of offspring with as many females as possible. There should only be enough male offspring to insure that all female offspring are mated and can produce the next generation of mostly females. Extra males are entirely superfluous, would compete with their developing sisters for fly nutrients, and would reduce the reproductive capacity of those sisters. Such a strategy should be selected for and should predominate.
2) The best “strategy” for the second wasp “superovipositing” in a fly puparium (foundress #2), would be to produce relatively more males, who could then sexually outcompete foundress #1’s male offspring for access both #1’s and #2s female offspring. Smaller males who consume less fly innards and develop faster could also help compensate for foundress #2’s late start.
3) A reasonable prediction of all of this might be the following. Each mated female wasp should inspect each new puparium she comes across. If she cannot detect evidence that the puparium has been oviposited, she should lay mostly female eggs. If she can determine that the puparium has been oviposited, she should lay a relatively higher proportion of male eggs. This would constitute an “evolutionarily stable strategy” or ESS, in that potentially disruptive alternative strategies, such as producing other sex ratios, should be selected against.
You are going to check all of this out using three pure stocks of jewel wasps. These are:
1) “Wild” – dull red-brown eyes
2) “Scarlet” – bright red eyes
3) “Oyster” – pearly gray eyes
You will conduct three experiments which will run concurrently, as outlined in the data sheet below. We will go over methods of anesthesia, typing, sexing, and handling of wasps prior to starting these experiments
I. MAINTAINING AND HANDLING JEWEL WASPS
1) You should already be familiar with the procedures for CO2 anesthetizing, handling and sexing jewel wasps. If necessary, review these procedures in the guide for laboratory #2.
2) This lab will require you to come in several days in a row to transfer wasps. Make sure that you clearly assign and schedule these responsibilities with your lab partners.
3) Make particular note of the fact that you will have to isolate at least 5-6 virgin females for each of two color variants several days before the formal lab period to start this lab. Your instructor will be maintaining the wasp stocks and will inform you when this needs to be done. Again, the procedures for doing isolating virgin females are described in the guide for laboratory #2.
4) You are responsible for correct wasp sex identification, correctly and diligently applied procedures, and the well-being of your wasps. It is essential that all lab groups take this very seriously. One set of bad data from an incorrectly or sloppily conducted experiment by a single group can corrupt the compiled data of the entire class.
II. FOUNDRESS SEQUENCE AND SEX RATIOS
Read ALL of the instructions below before starting.
For this experiment you will need stock bottles of two color variants, containing adult, mated females. The particular variants will be determined by the instructor. For the purposes of control and replicability your group will repeat this setup below with a total of 8 petri dishes – 4 with the wasp sequence A then B and 4 with the wasp sequence B then A.
1) Anesthetize wasps and separate out the females as needed for the following steps.
2) Place a single mature, pure-bred, mated female of type A in a covered petri dish with a single fly puparium and verify that she is viable and inspecting the puparium. Label the top of the dish with "Female 1 - [eye color A]"
3) 24 -36 hours later remove the female and replace her with a single pure-bred mated female of type B. Label the top of the dish again with "Female 2 - [eye color B]"
4) 48-72 hours later remove the second female.
5) About 2 weeks later, after the wasps have eclosed but before they have died of starvation, you must type, sex, and count all offspring which eclose and record this data on the data sheet.
Q1: What is your hypothesis for the relative sex ratios in offspring of the first wasp relative to the second wasp in each dish?
III. FOUNDRESS POPULATION DENSITY AND SEX RATIOS
Read ALL of the instructions below before starting.
For this experiment you will need stock bottles of only one color variant, containing adult, mated females. The particular variant will be determined by the instructor. For the purposes of control and replicability your group will repeat this setup below with a total of 8 Petri dishes – 2 with each number of foundress wasps.
1) Anesthetize wasps and separate out the females as needed for the following steps.
2) Place two fly puparia in each of four petri dishes.
3) Add female wasps to the dishes as follows:
dish 1 - 1 female wasp
dish 2 - 2 female wasps
dish 3 - 4 female wasps
dish 5 - 8 female wasps
4) Label the top of each dish with the number of foundress wasps added.
5) About 1 week later, but before any of the next generation of wasps eclose, remove all of the foundress female wasps.
6) About 2 weeks after starting the experiment after the new generation of wasps have eclosed, but before they die, you must sex and count all offspring in each dish and record this data on the data sheet.
Q2: What is your hypothesis for the relative sex ratios of offspring in each dish as the population of ovipositing female wasps increases?
IV . FOUNDRESS VIRGINITY AND PUPARIUM CHOICE
Read ALL of the instructions below before starting.
For this experiment you will need stock bottles of two color variants, containing adult, mated females. You will also need virgin females of both color variants. The particular variants will be determined by the instructor. For the purposes of control and replicability your group will repeat this setup below with a total of 8 Petri dishes – 4 with each color combination of mated and virgin female foundress wasps.
1) Anesthetize wasps and separate out the females as needed for the following steps.
2) Prepare your petri dish by drawing a line down the center on on the outside of the bottom of the dish. Label one side "O" and the other side "NO", again on the outside of the bottom dish.
3) Place a single puparium on the "O" side, add a single mature, pure-bred, mated female of type A and cover the dish. Verify that the female is viable and inspecting the puparium. Label the top of the dish with "Mated Female 1 - [eye color A]"
4) 24-36 hours later remove the mated female. Make sure that the oviposited puparium is on the "O" side of the dish. Add one more fly puparium, which has been warmed to room temperature, to the "NO" side of the dish. Now add a single pure-bred virgin female of type B. Label the top of the dish again with "Virgin Female 2 - [eye color B]"
4) Observe the virgin female for at least 20 minutes. Record how much time she spends inspecting each puparium. If you plan this correctly, you can observe and behaviorally record from all eight petri dishes simultaneously.
5) 12 - 18 hours later remove the second female. Note: this will most likely be very early in the morning of the day after the designated lab period.
6) About 2 weeks later, after the wasps have eclosed but before they have died of starvation, you must type, sex, and count all offspring which eclose and record this data on the data sheet.
Q3: What is your hypothesis about which puparium the virgin female will preferentially oviposit?
Q4: From your behavioral observations can you make a guess as to how the female is inspecting the puparia - i.e. what sensory modality and information is she using to determine whether the puparium has been previously oviposited?
V . WRITE-UP FOR THIS LAB
Data for all three experiments will be collected and compiled across the four lab groups. You are responsible for having your data ready in a timely manner to contribute it to this group effort.
The write-up for this lab should include all three experiments. For each you should provide a brief introduction, a tabular and/or graphical presentation of the results, and a short discussion of what the results imply for offspring sex ratio determination in jewel wasps.