DOI: 10.5061/dryad.sqv9s4n5x
title: Data from: Exoskeleton ageing and its relation to longevity and fecundity in female Australian Leaf Insects (Phyllium monteithi)

description: 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.
description: Source
 and maintenance of <em>P.
 monteithi</em> Eleven <em>P.
 monteithi</em> 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 <em>Syzygium australe</em>
 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 <em>S. australe</em> 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 &lt;
 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 &lt; 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 <em>S.
 australe</em> 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 <em>P. monteithi
 </em>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
 <em>lmer </em>function in the lme4 package (Bates et al. 2015)
 in R version 3.5.1 (R_Core_Team 2013). Fixed effects were tested using
 <em>F</em>-tests based on Satterthwaite’s degrees of freedom
 using the lmerTest package (Kuznetsova et al. 2017). We
 used the <em>lm </em>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.