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Data from: Exoskeleton ageing and its relation to longevity and fecundity in female Australian Leaf Insects (Phyllium monteithi)

Citation

Bonduriansky, Russell; Creak, Caitlin (2022), Data from: Exoskeleton ageing and its relation to longevity and fecundity in female Australian Leaf Insects (Phyllium monteithi), Dryad, Dataset, https://doi.org/10.5061/dryad.sqv9s4n5x

Abstract

Senescence is a decline in reproduction and survival rate with advancing age resulting from deterioration of somatic tissues and systems throughout the body. Age-related somatic changes (somatic ageing) have been studied extensively in vertebrates but are less well known in other animals, including insects. Since adult insects have very limited ability to repair their exoskeleton, somatic ageing could involve deterioration and discolouration of the cuticle. We investigated age-related changes in wing pigmentation and abdominal cuticle necrosis in females of the Australian leaf insect Phyllium monteithi. Adult females varied markedly in the extent and pattern of pigmentation on their bodies, and we found that pigment spots on the forewings increased in size with age in most individuals. As females aged, most individuals also exhibited increasing levels of abdominal cuticle necrosis, resulting in the loss of abdominal cuticle along the margin of the abdomen. Neither the extent of pigmentation nor cuticle loss were clearly associated with reduced fecundity or longevity in the protected laboratory environment, but it remains unknown whether these age-related changes have functional implications in the wild. Our results show that the P. monteithi exoskeleton undergoes complex changes with age, with potential implications for functional traits and fitness.

Methods

Source and maintenance of P. monteithi

Eleven P. monteithi females (including adults and late-instar nymphs) were purchased from a breeder (Australian Insect Farm, Innisfail, Queensland; Jack and Sue Hasenpusch, proprietors) in April 2017. These individuals came from a stock originally derived from a small number of females collected at Kuranda and one male collected at Garradunga, Queensland, and propagated for several generations through a mix of sexual and parthenogenetic reproduction (Jack Hasenpusch, personal communication). It is not known precisely how these eleven females were related, or whether they developed from fertilized or unfertilized eggs. The insects were provided with live Syzygium australe plants as food, and sprayed daily with water. These females began to lay eggs on 1/05/2017 and ~ 100 eggs were collected and incubated in moist cocopeat at 27°C. Nymphs (N = 74) hatched from these eggs were transferred to 10 fresh S. australe plants (1.5 – 2 m tall) in the UNSW glasshouse in May 2018. When these nymphs began to moult into adults in November 2018, the plants and insects were transferred to a controlled-temperature room at 27°C. The plants were arranged with branches interlocking to allow insects to move freely between them. The room was fitted with grow-lights and the plants were sprayed daily with water. The adult moult date for each individual was recorded, and each adult was marked on one wing with a unique number using a permanent marker. A total of 40 adult females and two adult males were thus obtained. This sex ratio suggests that most of these individuals were produced via parthenogenesis (which typically results in all-female broods in phasmids), but that at least one of the females obtained from the breeder mated and laid fertilized eggs (which develop into both females and males). Both males died within a few days of their adult moult. The 40 females, which underwent their adult moult between the 28th of August 2018 and the 12th February 2019 were used to investigate somatic ageing as described below.

Quantification of pigmentation and cuticle necrosis

The 40 females were imaged up to three times over the course of their adult life at ages (days from adult moult) ranging from 1 to 148 days. Both dorsal and ventral images were made each time using a Sony RX10 camera. The first set of images were made for all 40 females (age 9 ± 22 d, range 1 to 108 d) old. Because several females died over the course of the study, second images were made for 36 females (age 69 ± 23 d, range 50 to 172 d), and third images were made for 30 females (age 91 ± 20 d, range 45 to 148 d). This broad range of adult moult dates and ages enabled us to investigate senescence of individual females while reducing confounding effects of timing and seasonality.

ImageJ software (Schneider et al. 2012) was used to quantify pigmentation (i.e., brown patch areas) from dorsal images (Fig. 2a-c) and abdominal cuticle necrosis from ventral images (Fig. 2d-f) at successive ages for each female. To estimate the relative sizes of brown patches, the margin of one wing was traced to obtain wing area (Fig. 2b), and then the margins of all brown patches on that wing were traced to obtain their areas (Fig. 2c). The proportion of total wing area covered by brown patches was calculated by dividing the total brown patch area by total wing area. Brown patches covered < 2% of wing surface in all individuals. 

To estimate the relative extent of abdominal cuticle necrosis, the total ventral surface area of the intact abdomen was obtained by tracing around the abdominal margin, with the abdominal contour extrapolated across any indentations in the margin (Fig. 2e). The margins of all indentations were then traced to obtain their areas (Fig. 2f). The proportion of abdominal area lost to cuticle necrosis was then calculated by dividing the summed areas of the indentations by the total area of the intact abdomen. The proportion of the cuticle lost was < 1% in all cases, except for three individuals that showed signs of abdominal damage caused by other individuals chewing on the margins of their abdomen. Data from images where such damage was apparent (N = 4 images from 3 females) were excluded from analysis.

Wing length in mm was measured for each female as an index of body size.

Quantification of longevity and fecundity

Fecundity was estimated by transferring each female to an individual container (20 cm wide x 40 cm high) with a mesh lid and collecting eggs over 14 days. Containers were sprayed daily with water, and fresh S. australe branches (inserted in 250 mL containers filled with water) were provided every few days for food. Females were 38 ± 15 days old at the start of egg collection (range 31 to 111 d).

The plants on which focal females were feeding were inspected daily and dates of death were recorded. In addition, we censused the population on six occasions at intervals of approximately two weeks, attempting each time to locate each live individual and bodies of any missing individuals. Nonetheless, because P. monteithi females are extremely cryptic on their host plants, lifespan could not be determined with accuracy for 20 females. For these individuals, the ages at final sighting were included in the lifespan analyses as censored data.

Statistical analysis

We investigated the effect of age on pigmentation (i.e., the proportion of the wing covered by brown spots) and cuticle necrosis (i.e., the proportion of cuticle lost) by fitting separate Gaussian mixed models of these response variables, with female age, body size (wing length) and moult date as fixed effects and female identity as a random effect to account for repeated measures of individual females. Because the response variables were proportions, they were arcsine-transformed for the analysis. These models were fitted using the lmer function in the lme4 package (Bates et al. 2015) in R version 3.5.1 (R_Core_Team 2013). Fixed effects were tested using F-tests based on Satterthwaite’s degrees of freedom using the lmerTest package (Kuznetsova et al. 2017).

We used the lm function in base R to test for effects of female age at the start of egg collection, moult date, and wing length as fixed effects on female fecundity (eggs laid per day). To determine whether increased pigmentation or increased abdominal cuticle loss were associated with reduced fecundity, wing pigmentation (mean across all ages) and cuticle necrosis (mean across all ages) were then added to the model of female fecundity.

We used the coxph function in the Survival package (Therneau 2021) in R to test for effects of moult date and wing length on hazard (mortality) rate. To determine whether increased pigmentation or increased cuticle necrosis were associated with reduced longevity, these variables were then added to the cox model of female hazard rate.

Funding

Australian Research Council, Award: DP170102449