Use of X-ray micro-computed tomography to study the moult cycle of the freshwater amphipod Gammarus pulex
Introduction
The moult cycle, which is under endocrine control, is essential for crustacean growth affecting both the outer integument and internal organs. Trevisan et al. (2014) showed that moulting individuals stop feeding, and thus the process interferes with the digestive tract. It is thus critical that experimental studies of crustacean physiology or their response to abiotic or biotic stressors consider the stage an organism is at in their moult cycle at the point of study.
Several studies have divided the moult cycle of crustaceans into 5 basic stages: A, B, C, D1 and D2 (Cornet et al., 2012; Trevisan et al., 2014). The classification by Trevisan et al. (2014) is based on external morphological features such as body colour, presence or absence of gut contents and red-orange lipid storage droplets along the posterior borders of the tergites and coxal plates, as well as eye colour changes. They describe that just after the moult (stage A), the exoskeleton was soft and the specimen a greyish yellow colour with few lateral red-orange dots of stored lipids and predominantly white eyes sometimes speckled with black dots. Specimens in late post-ecdysis (stage B) were described as greyish to greenish colour with blackening of the eyes speckled with white spots, but no remaining red-orange lateral lipid dots. Very late post-ecdysial to anecdysial specimens (stage C) are described as having a fully rigid cuticle that appeared greenish coloured with predominantly black eyes although occasionally still flecked with white spots. Early pre-ecdysial specimens (stage D1) had completely black eyes, were olive green in colour with many obvious rows of red-orange dots along the posterior border of the tergites and coxal plates. Finally, late pre-ecdysial specimens (stage D2) had a yellowish-orange colour and even better developed red-orange lateral dots. Because the cuticle is relatively thin throughout the entire moult cycle the presence or absence of food in the digestive tract was easy to observe.
Other classification systems exist whereby stage C may be further divided into 4 separate stages, and stage D subdivided into as many as 7 or more individual stages (Drach, 1939; Drach and Tchergonovtzeff, 1967; Graf, 1986). Essentially, the pre-ecdysial stage D starts with the secretion of enzyme-containing ecdysial droplets (stage D0) which begin to gradually dissolve away and soften the inner part of the old cuticle. This process starts to free the epidermis from the old overlying cuticle, a process called apolysis, producing an ecdysial cleft (stage D1). Apolysis on the 3rd dactyl of amphipods has been described to occur in stage D (Cornet et al., 2012); as well as in stage C2, with further primitive matrix retraction in the dactylopodite throughout periods C3 and C4 (Graf, 1986). The progressive apolysis in the 3rd dactyl protopodite throughout stage D has been used to subdivide this moulting stage into 7 (Graf, 1986) or 5 (Cornet et al., 2012) separate stages. At the same time the epidermis starts to secrete a new cuticle consisting of an epicuticle and an exocuticle that grows in thickness throughout stages D2 to D4. Meanwhile the old cuticle thins and eventually is shed in the process of ecdysis. The post–ecdysial period is made up of stages A, B and C. Essentially the post-ecdysial period consolidates the new cuticle by secretion of the endocuticle and hardening and thickening of the exocuticle by a combination of mineralisation and sclerotization.
We have previously used X-ray micro-computed (micro-CT) tomography techniques to study various aspects of insect anatomy and physiology (Bell et al., 2012; Greco et al., 2012; Greco et al., 2014; Thielens et al., 2018), including the effect of cadmium on the Malpighian tubules of the seven spotted ladybird (Bell et al., 2012). The current study explored the suitability of this technique to determine the moult stage in a crustacean. Micro-CT identifies changes in the density of radiologically opaque materials such as the degree of calcification of exoskeleton and internal structures. Due to the demands of exoskeleton mineralisation, the calcium requirement will vary depending on the moult cycle stage in crustaceans (see for instance Greenaway, 1985, Wheatley, 1999), and thus has the potential to be a useful tool for moult stage classification. In Gammarus pulex there is evidence to suggest that the organism loses about 42% of body calcium into solution over a 2-3-day period preceding the moult and a further 54% is lost with the exuviae, leaving only about 4% in the newly moulted animal (Wright, 1980). The evidence from the measurement of calcium levels in different tissues including the chitinous exoskeleton and haemolymph at various moult stages and salinities in Litopenaeus vannamei (Chun-Huei and Sha-Yen, 2012) would suggest a progressive increase in cuticle calcium concentrations from stages A to mid stage D2 and then a small fall at stage D4 just before ecdysis. During the moult, G. pulex specimens shed not only their external exoskeleton, but also the ectodermal lining of their fore gut and hind gut (McLaughin, 1983). Decapods and amphipods have a gastric mill lining their stomachs to aid food mastication and digestion (Icely and Nott, 1992; Schmitz, 1992). The gastric mill consists of a series of gastric ossicles made up of thickened cuticle in the stomach lining which then may or may not be mineralised. Prior to ecdysis it is necessary for calcified gastric ossicles to be shed and dissolved in the animal’s gut. Thus, a micro-CT scan of a newly moulted stage A G. pulex would be likely to have little or no evidence of calcification of either its exoskeleton or gastric mill. Teleologically the gastric mill, like the exoskeleton, would need to mineralise quickly in stage B and early stage C. During pre-ecdysis, as apolysis took place progressively, at some stage late in stage D, radiological changes should also become apparent in the gastric mill as it became increasingly demineralised prior to moulting.
We hypothesise that because micro-CT identifies differences in radiologically opaque material, such as calcified structures, it will be a useful tool to identify the stage of the moult cycle in G. pulex. Thus, the aim was to compare moult stage derivation using micro-CT radiological criteria to the existing established techniques, e.g. 3rd dactyl histology and whole body and eye appearance (Cornet et al., 2012; Trevisan et al., 2014) to ascertain the validity of our novel micro-CT scanning method.
Section snippets
Gammarus pulex husbandry and processing
Adult Gammarus pulex specimens were collected from the river Cray, Orpington, Kent (51°23'08.8"N 0°06'32.0"E). G. pulex were kept at 14 °C with a natural light cycle and fed leaves collected from the river Cray, and gradually acclimatised over 1 week by replacing ½ of the river water every other day with clean artificial freshwater (AFW) based on the OECD 203 acute toxicity test water with a final salt concentration of 2 mM CaCl2; 0.5 mM MgSO4; 0.8 mM NaHCO3, 77.1 μM KCl, with a measured pH
Radiological assessment overview
In all 80 scanned specimens visualisation of the external features, as well as internal features such as the gastric mill, radio-opaque material in the gastrointestinal tract were obtained and used to assign to the various stages of moult A, B, C, D and late D (Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7, Fig. 8). Visualisation in the 65 specimens scanned at a setting of 40 μA and 98 kV was clearer than in the first 15 scanned at a setting of 61 μA and 73 kV.
Gammarus pulex - anatomical studies
Of the 80 scanned
Discussion
The study shows for the first time that X-ray micro-CT techniques can be used to determine the moult cycle stage of the freshwater amphipod Gammarus pulex. The moult cycle of G. pulex is described as lasting from about 15 days (Trevisan et al., 2014) to as many as 30 days (Cornet et al., 2012). These differences in reported moult cycle duration may reflect the fact that the G. pulex specimens examined by Trevisan et al. (2014) had a body length of 5 to 8 mm (measured from rostrum to the base of
Conclusion
G. pulex are ecologically important, recycling nutrients through leaf shredding and being a prey item for other organisms. Consequently, they are vital for the functioning of streams and rivers. It has been shown that an increasing number of pollutants may affect moulting as well as reproduction in these amphipods (Gismondi and Thome, 2014). Micro-CT scanning enables the moult cycle to be monitored, as well as at the same time being able to visualise integument calcification, internal
Declaration of Competing Interest
The authors report no declarations of interest.
References (21)
- et al.
Effects of two PBDE congeners on the moulting enzymes of the freshwater amphipod Gammarus pulex
Environ. Pollut.
(2014) - et al.
A preliminary report on the use of bench top micro-computerised tomography to study the malpighian tubules of the overwintering seven spotted ladybird Coccinella septempunctata L. (Coleoptera: Coccinellidae)
Psyche
(2012) - et al.
Fine structure and mineralization of the gastric mill in the crayfish Procambarus clarkii during the intermoult stage
J. Crust. Biol.
(2003) - et al.
Variation of calcium levels in the tissues and hemolymph of Litopenaeus vannamei at various moulting stages and salinities
J. Crust. Biol.
(2012) - et al.
Influence of female moulting status on pairing decisions and size-assortative mating in amphipods
J. Zool.
(2012) Mue et cycle d’intermue chez les crustaces Decapodes
Annales de l’Institut Oceanographiquie
(1939)- et al.
Sur la methode de determination des stades d’intermue et son application generale aux crustaces
Vie et Milieu. Serie A. Biologie Marine
(1967) Fine determination of the moult cycle stages in Orchestia cavimana Heller (Crustacea: Amphipoda)
J. Crust. Biol.
(1986)- et al.
Premolt calcium secretion in midgut posterior ceca in a terrestrial crustacean, Orchestia cavimana – ultrastructural changes in the postexuvial epithelium
J. Morphol.
(1983) - et al.
Calcium reabsorption in the posterior ceca of the midgut in a terrestrial crustacean, Orchestia cavimana-ultrastructural changes in the postexuvial epithelium
Cell Tissue Res.
(1985)
Cited by (3)
The tracheal system of the Common Wasp (Vespula vulgaris) – A micro-CT study
2023, Journal of Insect PhysiologyAn X-ray micro-computer tomography study of the Malpighian tubules of the Blue Bottle Blow Fly (Calliphora vomitoria) Diptera: Calliphoridae
2021, ZoologyCitation Excerpt :One insect, the fruit fly Drosophila melanogaster (D. melanogaster) may prove to be a useful model for studying human renal tract development (Davies et al., 2019), nephrolithisis and urolithiais (Chen et al., 2011, 2012) with the aid of X-ray Micro-Computer Tomography (MicroCT) techniques, similar to using non-contrast enhanced abdominal Computer Tomography (CT) for detecting renal pathology, including calculi, in human patients (Chen et al., 2019). Building on the study of radiological delineation of the calcium oxalate concretions’ or “urolithiasis” in the D. melanogaster Malpighian tubule by Chen et al., 2019, we endeavoured to use MicroCT as in Bell et al., 2020 to discern whether the blue bottle blow fly, Calliphora vomitoria (C. vomitoria) would prove a more effective model than D. melanogaster for radiologically studying both renal tract development and urolithiasis. We reasoned that both C. vomitoria and their close relatives Calliphora vicina (C. vicina) are a) larger than D. melanogaster, which should enable better diagnostic abilities due to more distinguishable spatial resolution on anatomical images, b) have been studied in great detail for over 100 years (Lowne, 1892, Lowne, 1894; Perez, 1910) and Fig. 1, c) have been extensively used by forensic practitioners to estimate time of death in humans and other animals (Greenberg, 1991; Rutty et al., 2013; Brown et al., 2015) and furthermore d) a series of highly detailed papers on C. vicina have been published by the Natural History Museum of London specifically on the use of MicroCT to study the various stages of fly intrapuparial developmental (Richards et al., 2012; Martín-Vega et al., 2016; Hall et al., 2017; Martín-Vega et al., 2017).