DOI: 10.5061/dryad.mw6m905zw
title: Sperm limitation produces male biased family sex ratios

description: 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.
description: We used the scarlet eye strain of <em>N.
 vitripennis</em> (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 F<sub>1</sub> generation.
 Each day, wasps received ad libitum sugar-water on a cotton
 swab. We removed F<sub>1</sub> 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 F<sub>2</sub> male and female
 wasp pupae in these hosts. We only included the broods of
 F<sub>1</sub> 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.
description: The dataset from Holditch et al. (2022, attached) contains brood
 data from an oviposition series produced by 12 different <em>Nasonia
 vitripennis </em><em>females</em>.
 We report the total number of male and female offspring and sex ratio for
 each of six host pupae (<em>Sarcophaga bullata</em>), 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
 &gt;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.