Final Molt Research Articles

Growth patterns of the invasive crab Cronius ruber (Brachyura, Portunidae) in the Canary Islands (Central-East Atlantic)

The growth pattern, including the moult process, relative growth, and estimation of von Bertalanffy growth parameters of the invasive non-indigenus species (NIS) Cronius ruber were studied in the Canary Islands. Despite being found in both, native and invaded areas, the life cycle and ecology of this species remain unknown. The study revealed a greater abundance of females than males, with males exhibiting larger carapace sizes and weights than females. Throughout ontogeny, relative growth was observed, with males reaching carapace widths (CW) between 54.5 and 57.8 mm and females reaching 49.9 mm CW. Allometry in the growth of the males’ chelae and females’ abdomen was consistently positive. Cronius ruber was found to be a fast-growing species, reaching the postpubertal growth phase (maturity) between 0.5 and 1 year of age in males and between 0.4 and 0.8 years in females. The intermoult period increased by more than 638 % in males and 443 % in females with age. Individuals undergo 13 moults during the first phase of life (until 30 mm CW) and then reduced to 6 moults until the end of life, concluding with a final terminal moult. The lifespan until the terminal moult is approximately 2.5 years, followed by a survival time over 1.5 years. von Bertalanffy growth parameters estimated using monthly CW-frequency data for males (L∞=97.4 mm CW, k=1.89 years−1, to=0.31 years), and for females (L∞=86.72 mm CW, k=1.91 years−1, to=0.29 years). Similar results were obtained using growth increment data.

Sperm storage organs change with female age and mating history in the Golden Silk spider Trichonephila clavipes (Araneae: Araneidae)

AbstractFemale sperm storage organs in arthropods are used as taxonomic characters since it is assumed that they do not change after maturation. However, in the Golden Silk spider, Trichonephila clavipes, the shape and sclerotization of the spermathecae change with a mating event. Moreover, mating in T. clavipes consists of several hundred copulations, but the male transfers all sperm during the first insertion of each pedipalp. Given this puzzling mating behaviour, we hypothesize that multiple copulations serve purposes other than sperm transfer and uptake, such as further altering the morphology of the storage site and/or transferring substances other than sperm. We compared virgin female T. clavipes with females after two copulations, one for each spermatheca, and after ad‐libitum mating. Each treatment consisted of two sub‐treatments in which the state of the sperm storage organs was inspected after a short (1–3 days) or a long (11 days) time interval after the female's final moult. The size of the spermathecae increased twofold in all mating treatments compared to virgin females. The thickness of the spermathecal wall increased significantly with time in all treatments similarly. The spermathecal epithelium, which releases secretion into the lumen of the spermathecae, seems most active in virgin females during the time when mating would normally occur and turns less active after mating and with age. The spermathecae contain secretion produced by the female before mating; and after mating, sperm and another secretion transferred by the male. The numerous sperm‐less matings in the ad‐libitum treatment resulted in male secretion in the copulatory ducts of the female genitalia, likely impeding sperm transfer by subsequent males. Out study shows that female genitalia can change considerably after having reached maturity and suggests that sperm‐less matings evolved in the context of sperm competition.

Plastic brain structure changes associated with the division of labor and aging in termites.

Division of labor is a prominent feature of social insect societies, where different castes engage in different specialized tasks. As brain differences are associated with behavioral differences, brain anatomy may be linked to caste polymorphism. Here, we show that termite brain morphology changes markedly with caste differentiation and age in the termite, Reticulitermes speratus. Brain morphology was shown to be associated with reproductive division of labor, with reproductive individuals (alates and neotenic reproductives) having larger brains than nonreproductives (workers and soldiers). Micro-computed tomography (CT) imaging and dissection observations showed that the king's brain morphology changed markedly with shrinkage of the optic lobes during their long life in the dark. Behavioral experiments showed that mature primary kings lose visual function as a result of optic lobe shrinkage. These results suggested that termites restructure their nervous systems to perform necessary tasks as they undergo caste differentiation, and that they also show flexible changes in brain morphology even after the final molt. This study showed that brain morphology in social insects is linked to caste and aging, and that the evolution of the division of labor is underpinned by the development of diverse neural systems for specialized tasks.

Modulation of the porcine intestinal microbiota in the course of Ascaris suum infection

BackgroundThe porcine roundworm Ascaris suum impairs feed conversion and weight gain, but its effects on intestinal microbiota remain largely unexplored.MethodsModulation of the intestinal microbiota was assessed in pigs that were infected once with 10,000 A. suum eggs and pigs that received a trickle infection (1000 eggs/day over 10 days), compared with a non-infected control group. Six pigs each were sacrificed per group at days 21, 35 and 49 post-infection (p.i.). Faecal samples taken weekly until slaughter and ingesta samples from different intestinal compartments were subjected to next-generation sequencing of the bacterial 16S rRNA gene.ResultsThe results revealed marked differences between the single- and the trickle-infected group. Single infection caused a remarkable but transient decrease in microbial diversity in the caecum, which was not observed in the trickle-infected group. However, an increase in short-chain fatty acid-producing genera in the caecum on day 21 p.i., which shifted to a decrease on day 35 p.i., was common to both groups, possibly related to changes in excretory–secretory products following the parasite’s final moult. Faecal microbial interaction networks were more similar between the single-infected and control group than the trickle-infected group. In addition, a lower degree of similarity over time indicated that A. suum trickle infection prevented microbiota stabilization.ConclusionsThese different patterns may have important implications regarding the comparability of experimental infections with natural scenarios characterized by continuous exposure, and should be confirmed by further studies.Graphical

Developmental Stages of Grassenema procaviae Petter, 1959 (Cosmocercoidea: Atractidae) Found in the Stomach of Cape Hyrax (Procavia capensis)

Morphological examination was made of the larval forms of Grassenema procaviae (Cosmocercoidea: Atractidae), an autoinfective and viviparous nematode parasite in the stomach of Cape hyrax (Procavia capensis). Three different larval stages (second-, third- and fourth-stages), and the adult stage were found among the worms collected at necropsy of 3 hosts, which were reared in a zoo in Japan. Molting phases between the larval stages and the final molt to the adult stage were also observed. It was considered that the gravid female delivers the second-stage larva, which develops to the adult stage through 3 molts. The cephalic structure was identical throughout the second to adult stages; all with transparent filaments extending from the mouth. Because starch grains were frequently found attached to the filaments and the worm intestinal lumen also contained starch grains ingested, the filaments were surmised to act as nutrient catchers.

Sex-specific development causes patterned emergence in Culex mosquito

In order to understand why adults of the Culex mosquito emerge in a manner that follows a well-defined mathematical pattern depending on the sex of the species, preliminary investigations were carried out to determine if sex-related differences exist in the rate of growth and development in the immature stages. Culex egg-rafts sampled from the natural breeding environment were hatched in the laboratory and a total of 583 first instar larvae were allowed to grow in isolation. The time taken for the isolated mosquitoes to molt into the next stage was recorded until the mosquitoes finally became adults. At the final molt, the sex of the mosquitoes was determined and assigned retrospectively to every molting stage. Of the entire isolated mosquito, 464 (220 males and 244 females) were able to reach maturity. We observed that the developmental process occurring in the immature mosquito is sex-specific between the fourth instar larva and pupa. This event gives male Culex approximately one-day advantage over females and explains why males always emerge first and dominate the first few days during the emergence period. The process also alters the pre-existing symmetry in the molting time distribution of the female mosquito. The outcome is a pattern that could be described as a logarithmic function of the emergence time in male Culex, and a power function of the emergence time in female Culex. We recommend an investigation of the other species of mosquito as well as other holometabolous insects to see if this observation can be reproduced.

Correlated sexual selection on male genitalia, copulatory performance and nuptial gifts in a bushcricket (Orthoptera: Tettigoniidae) indicated by allometric scaling

Abstract We adopt an allometric framework of scaling relationships for comparison between mating-related traits in the middle European bushcricket Roeseliana roeselii (Hagenbach, 1822). Eight characters, covering ontogenetic fitness (size traits; fixed at final moult), male condition (mass traits) and mating motivation (reproductive behaviours), were analysed in unrestricted matings and in matings involving genital manipulation. Shortening the male titillators had no effect on mating-related traits in males. However, titillators, known to be under sexual selection, scale hyperallometrically, with larger males possessing proportionally longer titillators, performing more titillator movements and exhibiting a reduced duration of copulation. Scaling was also hyperallometric for spermatophore mass, with larger males being heavier and transferring heavier nuptial gifts. Both titillator length and spermatophore mass might be condition-dependent indicators, because their variances were nearly twice as large those of body size or body mass. Mass traits were also dynamic, increasing by 11% for male body mass and 17% for spermatophore mass between the first and second matings. Sexual selection by female choice seems to favour larger trait size in the bushcricket R. roeselii, acting in concert on titillator length, intensity of titillator movements and spermatophore mass.

Functional morphology of immature mating in a widow spider

BackgroundMating generally occurs after individuals reach adulthood. In many arthropods including spiders, the adult stage is marked by a final moult after which the genitalia are fully developed and functional. In several widow spider species (genus Latrodectus), however, immature females may mate a few days before they moult to adulthood, i.e. in their late-subadult stage. While the “adult” mating typically results in cannibalism, males survive the “immature” mating. During both “immature” and “adult” matings, males leave parts of their paired copulatory organs within female genitalia, which may act as mating plugs. To study potential costs and benefits of the two mating tactics, we investigated female genital morphology of the brown widow spider, L. geometricus. Light microscopy, histology and micro-computed tomography of early-subadult, late-subadult and adult females were conducted to determine the overall pattern of genital maturation. We compared genitalia of mated late-subadult and adult females to reveal potential differences in the genitalic details that might indicate differential success in sperm transfer and different environments for sperm storage and sperm competition.ResultsWe found that the paired sperm storage organs (spermathecae) and copulatory ducts are developed already in late-subadult females and host sperm after immature mating. However, the thickness of the spermathecal cuticle and the staining of the secretions inside differ significantly between the late-subadult and adult females. In late-subadult females mating plugs were found with higher probability in both spermathecae compared to adult females.ConclusionsSperm transfer in matings with late-subadult females follows the same route as in matings with adult females. The observed differences in the secretions inside the spermathecae of adult and late-subadult females likely reflect different storage conditions for the transferred sperm which may lead to a disadvantage under sperm competition if the subadult female later re-mates with another male. However, since males mating with late-subadult females typically transfer sperm to both spermathecae they might benefit from numerical sperm competition as well as from monopolizing access to the female sperm storage organs. The assessment of re-mating probability and relative paternity will clarify the costs and benefits of the two mating tactics in light of these findings.

Notes on Morphology and Life History of Probstmayria gombensis (Nematoda: Cosmocercoidea: Atractidae), Parasitic in Eastern Chimpanzees, Pan troglodytes schweinfurthii, in Bulindi, Uganda.

Probstmayria gombensis File, 1976 (Nematoda: Cosmocercoidea: Atractidae) individuals discharged in the feces of eastern chimpanzees, Pan troglodytes schweinfurthii, in Bulindi, Uganda, were examined morphologically. Adults and fourth-stage larvae, all females, found in the feces, and the third-stage larvae excised from the uterus of the gravid females were described. By close observation of the molting worms, it was considered that the uterine third-stage larvae attain molting phase, and then are laid to become fourth-stage larvae. Nutrients required for larval development in the uterus seem to be supplied by the mother after the eggshell is formed. After some growth in the host intestine, the fourth-stage larvae undergo the final molt to the adult stage. The genital primordium was very small in the early fourth-stage larvae but rapidly developed with embryonation in the pre-molt and molting phases. Such precocity suggests parthenogenetic reproduction without insemination by males. This style may enhance rapid autoinfection in the host intestine under the condition of male worm scarcity. Several ellipsoidal pseudocoelomocytes with granules of unknown function were found ventral to the intestine of the adults, fourth-stage larvae, and third-stage larvae.

Hauling up a hefty meal: Long‐Jawed spider (Araneae, Tetragnathidae) uses silk lines to transport large prey vertically through the air in the absence of a web

AbstractThe most well‐known use of silk among spiders is the formation of webs to capture flying prey. However, spiders have evolved many different foraging strategies involving silk, including the capture and subsequent manipulate of prey prior to consumption. Herein, I report on the use of silk lines by a long‐jawed spider from the Tetragnatha genus to move a large prey item vertically through the air. Field observations revealed a long‐jawed spider attaching multiple silk lines across the body of an adult dragonfly that had recently emerged from its final moult and attaching these threads to overlying vegetation to incrementally haul the prey item from the surface of a waterbody. My observations suggest that some Tetragnatha spiders are able to move large prey items that are much heavier then themselves using a series of silk lines that allow lift to be accomplished in a gradual and controlled manner. This is an interesting finding, given that other individuals from the same population were using web structures to passively catch much smaller, flying prey. This indicates that some Tetragnatha spiders not only participate in the mobile pursuit of prey but that they possess multiple foraging strategies that may allow them to exploit a larger number of prey types.

Late postnaupliar development of the freshwater copepods Lovenula falicifera and Metadiaptomus colonialis (Calanoida: Diaptomidae) from South Africa.

The life cycle of calanoid copepods consists of eggs hatching into nauplii (6 stages) which then moult into copepodids (5 stages), followed by the final moult into the adult female and male. The family Diaptomidae contains two subfamilies, Diaptominae and Paradiaptominae, with paradiaptomids almost exclusively consisting of African taxa. The copepodid stages III, IV and V were described for some freshwater diaptomine genera (i.e., Eudiaptomus Kiefer, 1932, Aglaodiaptomus Light, 1938, Skistodiaptomus Light, 1939, Leptodiaptomus Light, 1938, Megadiaptomus Kiefer, 1936 and Diaptomus Westwood, 1836). Copepods collected from Turfloop Dam, South Africa, with a plankton net were fixed and preserved in 70% ethanol. Calanoid copepods were studied under stereo- and light microscopes, using the wooden slide technique and features drawn. Examined specimens were identified as the copepodid stages of two African species, Lovenula falcifera (Lovén, 1845) and Metadiaptomus colonialis (van Douwe, 1914). Copepodids of the two species can be distinguished by their body size and the structure and size of the maxillipeds. The description and illustrations of three postnaupliar stages (CoIII, CoIV and CoV) are provided for both species. The identification of different stages is based on the number of urosomites, antennule development, the segmentation of legs 1-4, and the development of the fifth leg. These copepodids are compared with those of other described diaptomid genera.

Winner and loser effects of juvenile cricket Gryllus bimaculatus

Agonistic encounters of juvenile male crickets were analyzed behaviourally. In a pairing between adult and juvenile male crickets, the juvenile crickets were usually beaten by the adult males of over 3 days after their final moult. Juveniles, by contrast, won significantly more bouts against young adult males 2 days after their final moult. These findings are good indicators to predict which cricket will defeat which opponent. To examine the effect of previous social experience, two juvenile animals were paired first and then juveniles that became subordinate were paired with day 2 adults, while juveniles that became dominant were paired with day 3 adults 5 min after first pairing. Subordinate juveniles were beaten by day 2 adults, while dominant juveniles tended to win against day 3 adults. This is the first time that winner and loser effects have been demonstrated in juvenile crickets. Similar pairings with day 2 or day 3 adult males were performed 2 h after first pairings. Subordinate juveniles were still beaten by day 2 adults, while the winning rate of dominant juveniles against day 3 adults was decreased significantly. These results suggest that the retention time of loser effect lasted more than 2 h while that of the winner effect disappeared within a shorter period.

Contribution to understanding the evolution of holometaboly: transformation of internal head structures during the metamorphosis in the green lacewing Chrysopa pallens (Neuroptera: Chrysopidae)

BackgroundMetamorphosis remains one of the most complicated and poorly understood processes in insects. This is particularly so for the very dynamic transformations that take place within the pupal sheath of holometabolous insects. Only few studies address these transformations especially with regard to cranial structures of those holometabolous species where the larval and adult forms have a similar diet. It thus remains unclear to what extent the internal structures undergo histolysis and rebuilding. Here, the development of the brain and skeleto-muscular system of the head of Chrysopa pallens (Rambur, 1838) is studied. This species is a predator of aphids in the larval and adult stage.ResultsWe used micro-computed-tomography (μ-CT) to study the transformations of the larval, prepupal and pupal head within the cocoon. We first assessed the morphological differences and similarities between the stages. We then determined the point in time when the compound eyes appear and describe the re-orientation of the head capsule which transforms the prognathous larva into a hypognathous adult. The internal head muscles are distinctly more slender in larvae than adults. In addition, the adults have a significantly larger brain which is likely needed for the processing of the signals obtained by the adults vastly expanded sensory organs that are presumably needed for dispersal and mating. Our study shows that the histolysis and modification of the inner muscles and skeletal elements take place within the prepupa. The central nervous system persists throughout metamorphosis but its morphology changes significantly.ConclusionOur study reveals that not only the inner structures, but also the outer morphology continues to change after the final larval moult. The adult cuticle and internal structures form gradually within the cocoon. The histolysis and rebuilding begin with the skeletal elements and is followed by changes in the central nervous system before it concludes with modifications of the musculature. This order of events is likely ancestral for Holometabola because it is also known from Hymenoptera, Diptera, Mecoptera, and Coleoptera.

Chronic and Acute Oral Administration of Caffeine Enhance Performance of Olfactory Learning in Crickets

Crickets were arbitrarily fed a caffeine-containing diet during their lifetime or were orally administered caffeine in solution once during the conditioning session. These chronically or acutely treated crickets were used to test whether there were positive effects of caffeine on their learning performance. The effects of chronic exposure to caffeine on crickets' growth were also examined by measuring their body weight and counting the number of adults that emerged from larvae. Chronic treatment with relatively high concentrations of caffeine (5 mg/g and 0.5 mg/g in the diet) impaired the growth of crickets. Mortality was higher with 5 mg/g caffeine, and the survivors could not attain the final molt (eclosion). Body weight gain and eclosion rate were also negatively influenced in the group fed 0.5 mg/g caffeine. The olfactory learning paradigm was used to examine the effects of chronic caffeine treatment. Of the caffeine concentrations tested (0.5 µg/g to 0.5 mg/g), significant improvement in long-term memory (LTM) formation was observed only with 50 µg/g caffeine. Acute effects on olfactory learning were examined after oral administration of 0.1 nM to 10 µM caffeine solutions during the conditioning session. Administration of caffeine ≥ 1 nM consistently resulted in a significant improvement in LTM formation. These results suggest that chronic exposure to caffeine enhances learning performance in crickets at a specific dose (50 µg/g in the diet), although it inhibits their growth at higher doses (≥ 0.5 mg/g). In contrast, acute oral administration of caffeine in naive crickets may enhance their learning performance even at a very low (nanomolar) concentration.

E93-depleted adult insects preserve the prothoracic gland and molt again.

Insect metamorphosis originated around the middle Devonian, associated with the innovation of the final molt; this occurs after histolysis of the prothoracic gland (PG; which produces the molting hormone) in the first days of adulthood. We previously hypothesized that transcription factor E93 is crucial in the emergence of metamorphosis, because it triggers metamorphosis in extant insects. This work on the cockroach Blattella germanica reveals that E93 also plays a crucial role in the histolysis of PG, which fits the above hypothesis. Previous studies have shown that the transcription factor FTZ-F1 is essential for PG histolysis. We have found that FTZ-F1 depletion towards the end of the final nymphal instar downregulates the expression of E93, whereas E93-depleted nymphs molt to adults that retain a functional PG. Interestingly, these adults are able to molt again, which is exceptional in insects. The study of insects able to molt again in the adult stage may reveal clues about how nymphal epidermal cells definitively become adult cells, and whether it is possible to reverse this process.

The metamorphosis of insects and their regulation

The metamorphosis of insects and their regulation

The innovation of the final moult and the origin of insect metamorphosis.

The three modes of insect postembryonic development are ametaboly, hemimetaboly and holometaboly, the latter being considered the only significant metamorphosis mode. However, the emergence of hemimetaboly, with the genuine innovation of the final moult, represents the origin of insect metamorphosis and a necessary step in the evolution of holometaboly. Hemimetaboly derives from ametaboly and might have appeared as a consequence of wing emergence in Pterygota, in the early Devonian. In extant insects, the final moult is mainly achieved through the degeneration of the prothoracic gland (PG), after the formation of the winged and reproductively competent adult stage. Metamorphosis, including the formation of the mature wings and the degeneration of the PG, is regulated by the MEKRE93 pathway, through which juvenile hormone precludes the adult morphogenesis by repressing the expression of transcription factor E93, which triggers this change. The MEKRE93 pathway appears conserved in extant metamorphosing insects, which suggest that this pathway was operative in the Pterygota last common ancestor. We propose that the final moult, and the consequent hemimetabolan metamorphosis, is a monophyletic innovation and that the role of E93 as a promoter of wing formation and the degeneration of the PG was mechanistically crucial for their emergence. This article is part of the theme issue 'The evolution of complete metamorphosis'.

Prepatterning of Papilio xuthus caterpillar camouflage is controlled by three homeobox genes: clawless, abdominal-A, and Abdominal-B.

Color patterns often function as camouflage to protect insects from predators. In most swallowtail butterflies, younger larvae mimic bird droppings but change their pattern to mimic their host plants during their final molt. This pattern change is determined during the early fourth instar by juvenile hormone (JH-sensitive period), but it remains unclear how the prepatterning process is controlled. Using Papilio xuthus larvae, we performed transcriptome comparisons to identify three camouflage pattern-associated homeobox genes [clawless, abdominal-A, and Abdominal-B (Abd-B)] that are up-regulated during the JH-sensitive period in a region-specific manner. Electroporation-mediated knockdown of each gene at the third instar caused loss or change of original fifth instar patterns, but not the fourth instar mimetic pattern, and knockdown of Abd-B after the JH-sensitive period had no effect on fifth instar patterns. These results indicate the role of these genes during the JH-sensitive period and in the control of the prepatterning gene network.

Cricket tympanal organ revisited: morphology, development and possible functions of the adult-specific chitin core beneath the anterior tympanal membrane.

Vertebrates and insects are phylogenetically separated by millions of years but have commonly developed tympanal membranes for efficiently converting airborne sound to mechanical oscillation in hearing. The tympanal organ of the field cricket Gryllus bimaculatus, spanning 200μm, is one of the smallest auditory organs among animals. It indirectly links to two tympana in the prothoracic tibia via tracheal vesicles. The anterior tympanal membrane is smaller and thicker than the posterior tympanal membrane and it is thought to have minor function as a sound receiver. Using differential labeling of sensory neurons/surrounding structures and three-dimensional reconstructions, we revealed that a shell-shaped chitin mass and associated tissues are hidden behind the anterior tympanal membrane. The mass, termed the epithelial core, is progressively enlarged by discharge of cylindrical chitin from epithelial cells that start to aggregate immediately after the final molt and it reaches a plateau in size after 6days. The core, bridging between the anterior tracheal vesicle and the fluid-filled chamber containing sensory neurons, is supported by a taut membrane, suggesting the possibility that anterior displacements of the anterior tracheal vesicle are converted into fluid motion via a lever action of the core. The epithelial core did not exist in tympanal organ homologs of meso- and metathoracic legs or of nymphal legs. Taken together, the findings suggest that the epithelial core, a potential functional homolog to mammalian ossicles, underlies fine sound frequency discrimination required for adult-specific sound communications.

Spittlebugs snorkel in cuckoo spit

In the lexicon of garden pests, spittlebugs (Philaenus spumarius) are probably one of the least offensive. Sipping sap destined for a plant's leaves, the tiny spittlebug nymphs do not linger long; yet, the foam that covers them – known euphemistically as cuckoo spit – drives gardeners wild. However, when Philip Matthews moved from Australia to Vancouver, Canada, the frothy blobs dotted across the city during spring transfixed him. ‘I'd never been anywhere where they were so abundant’, he laughs. He also realised that the froth-encrusted insects are essentially manufacturing their own aquatic environment. ‘Some of my previous research had been looking at aquatic insects carrying bubbles of air to breathe while underwater’, recalls Matthews. Which got him wondering: how could the bugs breathe while lathered in bubbles? Were they inhaling air trapped in the foam or poking their abdomens out of the frothy blanket to breathe?To answer these questions, undergraduate Kephra Beckett located cuckoo spit on shrubs in the University of British Columbia (UBC) campus and carefully poked a tiny fibre-optic oxygen sensor into the edge of a blob, recording the amount of oxygen as she painstakingly moved the sensor through the foam. ‘We never saw sub-atmospheric levels of oxygen in the centre of the spittle mass as you'd expect if the bug was breathing oxygen from the surrounding bubbles’, says Matthews. In addition, when Beckett measured the insects’ oxygen consumption in the lab, she could clearly see their abdomens protruding from the bubbles, unless they were startled. Then, they dived beneath the foam and their oxygen consumption ceased until they re-emerged minutes later.So the nymphs weren't using their foam bubbles as an aqualung, they were snorkelling. However, Matthews knew that during their final life stage, before metamorphosing into adults, spittlebugs produce a larger bubble chamber by popping the surrounding bubbles. Might they be consuming the oxygen in the enlarged chamber during their final moult? Collecting youngsters on the verge of the transition, complete with foliage residences, and returning to the lab, Beckett cautiously inserted the oxygen sensor into the bubble chamber and found that the oxygen concentration was about 10% lower than that of the external atmosphere, suggesting that they were breathing the oxygen within. Her colleague Anne Robertson also sandwiched young spittlebugs between a microscope slide and a coverslip, to find out if they could resort to breathing the air trapped in the foam, and saw the youngsters puncturing the bubbles with their abdomens. In addition, when she coated the microscope slide with an oxygen-sensitive dye, she could see the dye glow intensely as the oxygen declined. The tiny nymphs are able to use their bubbles as an aqualung when necessary.In addition, Beckett and Matthews were curious to find out just how hard it is for the nymphs to dine on dilute xylem sap. According to Matthews, the sap is under negative pressure – air gets drawn into the sap channels when they are punctured – making it hard for spittlebugs to suck against. The question was, how hard? Back in the lab, Beckett recorded the carbon dioxide produced by the patiently waiting nymphs, before providing them with juicy sheep sorrel plants and rerecording their carbon dioxide production as they gorged on sap and spewed out foam. However, the insects' carbon dioxide production only increased by a modest 20%. ‘That was a big surprise’, says Matthews. ‘It's not much greater than their cost of walking’, he adds.So, spittlebugs appear not to be struggling to take a sip, but Matthews suspects that there may be more to the story and is looking forward to next spring when splodges of cuckoo spit begin bubbling up all over Vancouver again.