Haplo-diploid sex determination in the parasitoid wasp, Nasonia vitripennis (Walker), allows females to adjust their brood sex ratios. Females influence whether ova are fertilized, producing diploid females, or remain unfertilized, producing haploid males. Females appear to adjust their brood sex ratios to minimize “local mate competition,” i.e., competition among sons for mates. Because mating occurs between siblings, females may optimize mating opportunities for their offspring by producing only enough sons to inseminate daughters when ovipositing alone and producing more sons when superparasitism is likely. Although widely accepted, this hypothesis makes no assumptions about gamete limitation in either sex. Because sperm is used to produce daughters, repeated oviposition could reduce sperm supplies, causing females to produce more sons. In contrast, if egg-limited females produce smaller broods, they might use fewer sperm, making sperm limitation less likely. To investigate whether repeated oviposition and female fertility influence gamete limitation within females, we created two treatments of six mated female wasps, which received a series of six hosts at intervals of 24 or 48 hrs. All females produced at least one mixed-sex brood (63 total broods; 3,696 offspring). As expected, if females became sperm limited, in both treatments, brood sex ratios became increasingly male-biased with increasing host number. The interhost interval did not affect brood size, total offspring number or sex ratio, indicating females did not become egg limited. Our results support earlier studies showing sperm depletion affects sex allocation in N. vitripennis¸ and could limit adaptive sex ratio manipulation in these parasitoid wasps.

We used the scarlet eye strain of N. vitripennis (Ward’s Science, Rochester NY) in our experiments. Ward’s reports this strain to be derived from and occasionally outcrossed with wasps from outside sources, but once established in-house, their breeders tend to culture within the lineage, making the potential for inbreeding high. We note that our use of this strain could limit our conclusions, depending on how the selective environment these wasps experience with respect to mating and sperm storage differs from that found in nature. Given that extreme inbreeding via sib-mating immediately after eclosion is the sine qua non of parasitoid wasp mating systems, we considered this possibility an acceptable risk (see also Discussion).

Nevertheless, to minimize possible negative effects of inbreeding, to minimize maternal effects, and to standardize the relatedness among our experimental wasp stocks, we imposed two rounds of outbreeding before evaluating our results. Specifically, we isolated the Ward’s wasps as 12 d pupae and allowed them to emerge as adults at 25°C for approximately 48 hrs. Then we paired each parental generation (P) female with one haphazardly selected P generation male from a different host pupa for 18 h for mating. Following the mating period, we separated males from females, and provided each female with a host pupa for 24 hrs. We then removed these parasitized host pupae and stored them at 25C for 12 days to create our F₁ generation. Each day, wasps received ad libitum sugar-water on a cotton swab.

We removed F₁ wasp pupae from hosts and again created non-sibling male-female pairs for 18hrs (same conditions as above). We then isolated males from the presumably-mated females for 24 h. To determine whether repeated oviposition influenced female brood sex ratios, we allowed each mated female to oviposit on six hosts in succession. To determine whether differences in host availability may influence egg limitation in females, we introduced a new host to each mated female every 24 h (N = six females) or every 48 h (N = six females) for a total of six hosts per female. After 12 d of larval development at 25°C, we counted the number of F₂ male and female wasp pupae in these hosts. We only included the broods of F₁ females with at least one host that produced at least one son and one daughter in our analyses. To determine whether repeated oviposition caused females with higher fertility to deplete their supplies of stored sperm more than females with lower fertility, we recorded the total fertility of all females. 

We used Sarcophaga bullata pupae used as hosts in this experiment sourced from either Ward’s Science (Rochester NY) or from a colony we established from these pupae (following protocol in Werren and Loehlin 2009). All host pupae were stored at 5°C throughout the experiment. We combined commercial and stock pupae and distributed these hosts haphazardly to females.

The dataset from Holditch et al. (2022, attached) contains brood data from an oviposition series produced by 12 different Nasonia vitripennis females. We report the total number of male and female offspring and sex ratio for each of six host pupae (Sarcophaga bullata), which were distributed to mated female wasps at varying intervals (i.e., every 24 or 48 hrs).

The first row identifies information for each column. Each row contains data for a brood produced by a single female, on a single host. The interval at which females received unparasitized host pupae is given in Column 1 (i.e., 24 or 48 hrs). Individual females are distinguished by a lowercase letter in Column 2 (i.e., Female Identity). The six host pupae provided to females during the experiment are identified by Host Order (1-6) in Column 3.

Host pupae that females successfully parasitized (i.e., yielded >1 offspring) are identified in Column 4 under Host Number. The total number of male and female offspring counted in each host is given in Columns 5 and 6 (i.e., Nmales and Nfemales, respectively). Wasp offspring which could not be identified to either sex are counted in Column 7, (i.e., Nunknown). Brood size for each host is given in Column 8, and is the sum of Nmales, Nfemales, and Nunknown. Finally, Column 9 contains the brood sex ratio and is calculated as the proportion of males within the brood.