Testosterone identifies hatchling sex for Mojave desert tortoises (Gopherus agassizii).

The threatened Mojave desert tortoise (Gopherus agassizii) exhibits temperature-dependent sex determination, and individuals appear externally sexually monomorphic until sexual maturity. A non-surgical sex identification method that is suitable for a single in situ encounter with hatchlings is essential for minimizing handling of wild animals. We tested (1) whether plasma testosterone quantified by enzyme-linked immunosorbent assay differentiated males from females in 0-3 month old captive hatchlings, and (2) whether an injection of follicle-stimulating hormone (FSH) differentially elevates testosterone in male hatchlings to aid in identifying sex. We validated sex by ceolioscopic (laparoscopic) surgery. We then fit the testosterone concentrations to lognormal distributions and identified the concentration below which individuals are more likely female, and above which individuals are more likely male. Using a parametric bootstrapping procedure, we estimated a 0.01-0.04% misidentification rate for naïve testosterone samples, and a 1.26-1.39% misidentification rate for challenged (post-FSH injection) testosterone samples. Quantification of plasma testosterone concentration from small volume (0.1 mL) blood samples appears to be a viable, highly accurate method to identify sex of 0-3 month old hatchlings and could be a valuable tool for conservation measures and investigation of trends and variation in sex ratios for in situ wild nests.

A comparison of non-surgical methods for sexing young gopher tortoises (Gopherus polyphemus).

Many turtle species have temperature-dependent sex determination (TSD), raising the prospect that climate change could impact population dynamics by altering sex ratios. Understanding how climate change will affect populations of animals with TSD requires a reliable and minimally invasive method of identifying the sexes of young individuals. This determination is challenging in many turtles, which often lack conspicuous external sexual dimorphism until years after hatching. Here, we explore four alternatives for sexing three age classes of captive-reared young gopher tortoises (Gopherus polyphemus), a terrestrial turtle of conservation concern native to the southeastern United States: (1) naive testosterone levels, (2) testosterone levels following a follicle stimulating hormone (FSH) challenge, (3) linear morphological measurements, and (4) geometric morphometrics. Unlike some other turtle species, male and female neonatal gopher tortoises have overlapping naive testosterone concentration distributions, justifying more complicated methods. We found that sex of neonates (<7 days old) is best predicted by a "random forest" machine learning model with naive testosterone levels and morphological measurements (8% out-of-bag error). Sex of hatchlings (4-8 months old) was predicted with 11% error using a simple threshold on naive testosterone levels, or with 4% error using a simple threshold on post-FSH testosterone levels. Sex of juveniles (approximately 3.5 years old) was perfectly predicted using a simple threshold on naive testosterone levels. Sexing hatchlings at >4 months of age is the easiest and most reliable non-surgical method for sex identification. Given access to a rearing facility and equipment to perform hormone assays, these methods have the potential to supplant laparoscopic surgery as the method of choice for sexing young gopher tortoises.

Matricide and queen sex allocation in a yellowjacket wasp.

In many colonies of social insects, the workers compete with each other and with the queen over the production of the colony's males. In some species of social bees and wasps with annual societies, this intra-colony conflict even results in matricide-the killing of the colony's irreplaceable queen by a daughter worker. In colonies with low effective paternity and high worker-worker relatedness, workers value worker-laid males more than queen-laid males, and thus may benefit from queen killing. Workers gain by eliminating the queen because she is a competing source of male eggs and actively inhibits worker reproduction through policing. However, matricide may be costly to workers if it reduces the production of valuable new queens and workers. Here, I test a theoretical prediction regarding the timing of matricide in a wasp, Dolichovespula arenaria, recently shown to have facultative matricide based on intra-colony relatedness. Using analyses of collected, mature colonies and a surgical manipulation preventing queens from laying female eggs, I show that workers do not preferentially kill queens who are only producing male eggs. Instead, workers sometimes kill queens laying valuable females, suggesting a high cost of matricide. Although matricide is common and typically occurs only in low-paternity colonies, it seems that workers sometimes pay substantial costs in this expression of conflict over male parentage.