Gastroliths and biomineralization related gene expression in crayfish

Amir Sagi

Department of Life Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel

National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer Sheva, Israel

Abstract: Having a rigid exoskeleton, crustacean growth processes demand a repetitive molting cycle. During the molt cycle crustaceans form their cuticular structures and many crustacean species, such as crayfish, develop functional structures such as gastroliths, in a specific timing, making them ideal structural biology study organisms. Gastroliths are calcium storage organs deposited on either side of the stomach, growing along the molt cycle, and collapsing into the stomach at ecdysis to serve as mineral sources for calcification of the exoskeleton after molting. The formation of such structures involves growth of the organic matrix including chitin and cuticular proteins, and mineral deposition. The process of gastrolith development in crayfish involves the construction of an extracellular chitinous scaffold over which mostly stabilized amorphous calcium carbonate is deposited. These structures involve a wide repertoire of structural and functional proteins including ion transporters. Based on a molt-related transcriptomic library from crayfish, cuticular and gastrolith protein encoding-genes were mined using a multigenic binary patterning approach developed specifically for the study of structural proteins in crustacean cuticle and gastroliths. Several proteins were studied with respect to cuticular structures and ion transport into exoskeletal biomineralization sites of calcium carbonate.

The gastroliths also serve as an indexed tool to monitor crayfish molt cycle through a Molt Mineralization Index (MMI). Using MMI values the molt cycle could be precisely timed and the molecular background behind growth and biomineralization could be studied. This way molt related transcriptomic libraries were constructed and functional genomics tools such as RNA interference (RNAi) and CRISPR applied to discover the function of relevant proteins. RNAi manipulations significantly affected molt cycle outputs suggesting important functions to several cuticular proteins and ion transporters. Precise functional genomic research and applications of genome editing will contribute to further understanding of the physiological background behind the extremely efficient biomineralization processes in crayfish.

2．フィレンツェ大学博物館　ギアナ・インセンティ博士　

　地中海域における外来十脚目甲殻類：3つのケーススタディ

（発表内容）海産の侵略的外来種は固有の生態系に大きな負の影響を与え、これが人間生活にも悪影響を与えることもあります。地中海に侵入した海産の外来十脚目のCallinectes sapidus、Charybdis longicollis、Percnon gibbesiを例ケーススタディ例として、地中海の在来生態系と経済面への影響についての検討例を紹介します。 

Alien Crustacea Decapoda in the Mediterranean Region: Three Case Studies

Gianna Innocenti

University Museum System, Natural History Museum, University of Florence, via Romana 17, 50125 Firenze (FI), Italy

Abstract: The introduction of non-native species (NNS) is a significant driver of biodiversity change. Some introductions can alter community dynamics, and lead to the displacement or reduction of native species, affecting the overall health and functioning of marine environments, i.e. the disruption of native ecosystems.

Spatial and temporal spread of NNS in the Mediterranean Sea advanced concurrently with successive enlargements of the Suez Canal, with range expansion of Atlantic species, and increases in mean seawater temperatures and in the prevalence, duration and severity of marine heat waves. The invasion poses a challenge for the environmental policies of the Mediterranean countries in terms of commitment towards conservation of biological diversity.

Eighty-eight non-native decapod species have been recorded in the Mediterranean. This presentation will examine the ecological and economic examples impacts of the portunid crabs Callinectes sapidus and Charybdis longicollis, and the percnid Percnon gibbesi.

3．フィレンツエ大学理学部　エレナ・トリカリコ博士

　欧州におけるアメリカザリガニ

（発表内容）ヨーロッパでも日本同様にアメリカザリガニが移入し、南ヨーロッパを中心に在来生態系に深刻な被害を与えています。その対策は各国で取り組まれていますが特に地中海に位置するイタリアでの事例を紹介します。 

The red swamp crayfish Procambarus clarkii in Europe

Elena Tricarico

Department of Biology, University of Florence

Via Madonna del Piano 6, 50019 Sesto Fiorentino (FI), Italy

Abstract: The red swamp crayfish Procambarus clarkii has been widely introduced in Europe for aquaculture and ornamental purposes since 1970s, establishing several abundant populations, especially in the southern countries. Italy is, indeed, one of the most affected countries by the species: P. clarkii was introduced in the 1980s and is now present in almost all the 20 Italian regions, main islands included. In Europe and Italy, besides being a vector of crayfish plague, which is responsible for large-scale disappearance of native crayfish species, it causes severe impacts on diverse aquatic ecosystems, due to its rapid life cycle, dispersal capacities, burrowing activities and high population densities. Most studies showed the decline of macrophytes and predation on several species (amphibians, molluscs, and macroinvertebrates), highlighting how this biodiversity loss leads to changes in ecosystem service. It can cause also important economic damages, inducing the collapse of banks thanks to its digging activity and impacting the rice production by consuming the young plants. For its ecological impacts, the species has been included in the list of Invasive Alien Species of Union concern linked to the EU Regulation 1143/2014, and its management is mandatory for the European Member States, Italy included. An integrated approach is recommended to tackle this species and several measures have been applied to control and reduce its populations. Some successful management projects will be illustrated during the presentation.

4．Crustacean molt physiology: phosphate transporter in crayfish biomineralization

甲殻類の脱皮生理：ザリガニのバイオミネラリゼーションにおけるリンの移動

Shai Avraham Shaked (1, 2) Simy Weil (1), Rivka Manor (1), Eliahu David Aflalo (1,3,) Jerry Eichler (1) & Amir Sagi (1, 2):  1 Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel. 2 The National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva, Israel. 3 Department of Life Sciences, Achva Academic College, Shikmim, Israel

Shai Avraham Shaked 

Abstract: Crustacean molt physiology is widely studied with processes ranging from developing a larger exoskeleton for growth to morphological changes in a new exoskeleton. The Redclaw crayfish Cherax quadricarinatus is used as a model animal to study the molt cycle, its physiology and key proteins involved in biomineralization of the exoskeleton. Like many other invertebrates, crustaceans mineralize their exoskeletons primarily by calcium carbonate in different polymorphs. C. quadricarinatus utilizes the stabile amorphous form of CaCO3 (ACC) in parts of its exoskeleton and gastroliths (transient calcium storage organs). The stabilization of amorphous calcium carbonate was thought to be controlled by phosphate ions (Pi), thus the role of phosphate transporters in crayfish molt cycle and mineralization was studied. A molt-related transcriptomic library and a novel binary patterning approach were exploited to reveal a SLC20A2 transport protein encoding gene in the crayfish C. quadricarinatus. Functional genomics experiments using RNAi demonstrated that Cq-SLC20A2 transport of phosphate to the mineralization site of amorphous calcium carbonate (ACC) at the exoskeleton. Following silencing, increased crystallinity of ACC was demonstrated at the gastroliths together with noted reduction in phosphate content (Fig. 1). This first discovery of the involvement of Cq-SLC20 in crustacean exoskeletal mineralization through phosphate active transport sheds light on long hypothesized putative role for Pi in CaCO3 biomineralization.

Figure 1. Pi content in gastroliths during the molt cycle following RNAi-induced loss-of-function of CqSLC20A2. Calcium and phosphate distribution in a representative control animal injected with dsGFP (left) and calcium and phosphate distribution in a representative animal of the silenced group injected with dsCqSLC20A2 (right). Purple – calcium. Red – Phosphate. 

5. Monosex prawns and crayfish as environmental biocontrol agents against pest snails

Sharon Moscovitz (1), Shai Avraham Shaked (1, 2), Hanin Wattad (1), Hanoch Glassner (3), Eliahu D. Aflalo (1, 4), Ofer Ovadia (1) & Amir Sagi (1,2): 1 Department of Life Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel. 2 National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer Sheva, Israel. 3 Eden Research Station, Beit She'an, Israel. 4 Department of Life Sciences, Achva Academic College, Shikmim, Israel

Sharon Moscovitz 

Abstract: Snail-borne diseases (SBD) and freshwater snails themselves are presenting a global challenge: Biomphalaria, Bulinus and Oncomelania transmit human Schistosoma; Thiaridae transmit various fish-infecting trematodes and Pomacea apple snails harm rice crops. These are world-wide economic, health and veterinary issues. The snails are often targeted by molluscicides, and lately the integration of more sustainable biocontrol agents was suggested. Here we present noninvasive monosex populations of prawns (Macrobrachium rosenbergii) and crayfish (Cherax quadricarinatus), that were generated using non-GMO methods to serve as biocontrol agents, by manipulation of the AIG-switch in early developmental stages of M. rosenbrgii, or by using naturally occurring intersex crayfish to produce all-female progenies for C. quadricarinatus.

We examined the predation abilities of prawns on four species of aquacultural pest SBD transmitting snails which are found together in the field: Melanoides tuberculata, Tarebia granifera, Thiara scabra and Pseudosuccinea columella. The prawn was previously proven a successful size-selective predator of M. tuberculata and T. scabra in the lab and field. In our following food-choice experiment, M. rosenbergii was successful in predating all four species, showing a preference for the soft-shelled snail first (P. columella) and the hard-shelled snail the least (T. scabra).

We subsequently examined the predation abilities of C. quadricarinatus against the hard-shelled species, and size-grade preferences by presenting small and medium snails. We found it a faster predator than the prawn, and very efficient with nearly 100% predation of the snails in both sizes.

We will discuss both predators as biocontrol agents in snail-struck areas and in agricultural polyculture, with the ability to choose the better suiting crustacean for the specific snail-control purpose. Furthermore, considering the requirements of biocontrol agents to be noninvasive themselves, we encourage strictly monosex crustacean populations as safely monitored biocontrol, and an additive crop on their own, as a win-win-win sustainable snail and SBD solution.

II. 招聘講演関連講演

6.絶滅危惧種ニホンザリガニの保全と環境DNAを用いた生息地特定

Conservation of Japanese crayfish and habitat identification using environmental DNA

飯塚凜人・佐藤尚貴・津谷桂名（秋田県立大館鳳鳴高等高校2年）

Rinto Iizuka & Naoki Sato & Keina Tsuya

Odate Homei High School

7.北海道のザリガニ保全方針の策定と今後の展開

Formulation and future development of freshwater crayfish conservation guideline in Hokkaido

山田浩行（パシフィックコンサルタンツ㈱）

Hiroyuki Yamada