Investigation of hatching and early post‐embryonic life of freshwater crayfish by in vitro culture, behavioral analysis, and light and electron microscopy

Abstract

The late embryonic and early post-embryonic life period of freshwater crayfish, which is the main time period of organogenesis, is poorly investigated because of the protective brooding behavior of crayfish mothers. A combination of in vitro culture, behavioral observations, and microscopic investigations of organs involved in hatching, attachment, exploration of the environment, and searching and processing of food yielded deeper insights in this important period of life. Experiments were performed with the robust parthenogenetic marbled crayfish. The following results were obtained: (1) Marbled crayfish can be raised in simple in vitro systems from 80% embryonic development to juvenile Stage 4 with up to 100% survival; (2) Hatching is prepared by chemical weakening of the egg shell and completed by levering actions of the hatchling's appendages; (3) The telson thread, a safety line that keeps the hatchling secured to the mother, is formed by secretions from the telson and the detaching inner layer of the egg case; (4) Molting Stage-1 juveniles are secured by an anal thread that results from delayed molting of the hindgut; (5) Active attachment of the hatchlings to the maternal pleopods with their 1st pereiopods is achieved by an innate fixed action pattern; (6) In vitro, juveniles are motile from Stage 2 despite incomplete development of their balance controlling statocysts. Movement pattern and social behavior vary greatly among individuals; and (7) Feeding starts in Stage 3, when the mouthparts and the gastric mill are fully developed. Onset of feeding is innate and does not require maternal contributions. In vitro culture of the isogenic marbled crayfish is recommended for broader use in research because it enables not only time and stage-specific sampling but also precisely timed experimental manipulations.

Production of different phenotypes from the same genotype in the same environment by developmental variation

“…For instance, the sternal artery, which connects the heart to the ventral thoraco-abdominal artery, can be either paired and bilateral symmetric, or unpaired and right or left asymmetric, as revealed by serial sectioning of more than 100 juveniles (Vogt et al, manuscript submitted for publication). Movement patterns and social behaviours were shown to vary considerably among stage-2 batch-mates raised from in vitro cultured eggs (Vogt, 2008). And the duration of embryonic development, hatching success, growth of the juveniles, and grade of malformation of the appendages varied considerably among sibs exposed in 12-well micro-plates to 100·g·l…”

The marbled crayfish (Decapoda: Cambaridae) represents an independent new species

“…Several important characteristics of marbled crayfish have been described in detail, including morphology (Kawai et al, 2009), embryonic development (Alwes & Scholtz, 2006;Seitz et al, 2005), life history (Seitz et al, 2005;Vogt, 2008;Vogt et al, 2004), parthenogenetic reproduction (Martin et al, 2007;Scholtz et al, 2003;Vogt et al, 2008) and a triploid karyotype . Using morphological characters and molecular markers, Martin et al identified the sexually reproducing slough crayfish Procambarus fallax from Florida as the closest relative of marbled crayfish and suggested the provisional name Procambarus fallax forma virginalis (Martin et al, 2010).…”

Symmetry variation in the heart‐descending artery system of the parthenogenetic marbled crayfish

“…It will be the challenge of future investigations to identify the epigenetic and molecular mechanisms that cause this symmetry variation. Because its embryos and early juveniles can be cultured in simple facilities, including microplates (Vogt, 2007(Vogt, , 2008a, the marbled crayfish may serve as an additional model to investigate the establishment and maintenance of bilateral symmetry and symmetry breaking in metazoans (Mercola and Levin, 2001;Palmer, 2004). Future studies using marbled crayfish raised under different experimental conditions could provide information on the modifiability of symmetry by the macroenvironment, and experiments with the sexually reproducing and closely related species, either Procambarus alleni or Procambarus fallax, could provide information on the genetic aspects of symmetry.…”

“…The parthenogenetic marbled crayfish, which was discovered in the mid 1990s (Scholtz et al, 2003;Vogt, 2008b), seems to be a particularly suitable candidate for a detailed investigation into this problem as it is the only known decapod crustacean to produce genetically identical offspring (Martin et al, 2007;Vogt et al, 2008). Moreover, this crayfish develops directly without metamorphosis Alwes and Scholtz, 2006) and can be reared in very simple environments including microplates (Vogt, 2007(Vogt, , 2008a. To examine whether symmetry variation in the descending artery of crayfish is the result of developmental variation we serial-sectioned isogenic embryos and early juveniles of marbled crayfish raised under identical environmental conditions.…”