Journal of Nematology 49(4):390–395. 2017. Ó The Society of Nematologists 2017. First Report of Matricidal Hatching in Bursaphelenchus xylophilus ADELA ABELLEIRA,ALICIA PRADO,ANDREA ABELLEIRA-SANMARTI´N, AND PEDRO MANSILLA Abstract: The reproductive strategy of the pinewood nematode (PWN), Bursaphelenchus xylophilus, is sexual amphimictic and oviparous. The incidence of intrauterine egg development and hatching in plant-parasitic nematodes is not a very common phe- nomenon. During the process of maintaining and breeding a B. xylophilus population isolated in Spain under laboratory conditions, evidence of matricidal hatching was observed. This is the first described case of this phenomenon in this species. Key words: Bursaphelenchus xylophilus, detection, endotokia matricida, laboratory conditions, matricidal hatching, morphology, nematode reproduction, physiology, pinewood nematode. Pine wilt disease, caused by the PWN B. xylophilus of B. xylophilus, we selected the breeding process on (Steiner et Buhrer) Nickle, is a major disease affecting potato dextrose agar (PDA) petri plates with B. cinerea. conifer forests and resulting in significant economic Nematode multiplication losses (Mota et al., 2009). At the beginning of the 20th century, the disease was especially prevalent in East A culture of B. cinerea in PDA plates was used to ob- Asian countries; however, at the end of the century, the tain nematode growth media in petri plates measuring disease started to spread toward Europe and currently 55 and 90 mm in diameter and incubated at 258C for 2 only affects the Iberian Peninsula, Portugal (Mota et al., and 7 d, respectively. Once proper growth of the fungus 1999), and Spain (Abelleira et al., 2011; Robertson had been verified, approximately 20 adult specimens of et al., 2011). In Galicia, one of the Spanish regions in B. xylophilus, mostly females, were placed on petri plates which this nematode has been identified, a B. xylophilus (55 mm in diameter). To multiply this original population, population was isolated and reared under laboratory the plates were incubated at 258C for 1 wk. Then, once the conditions with the aim of conducting further studies first population had multiplied, two pieces of medium, on its biology, pathogenicity, and control. Maintenance with a density of more than 100 nematodes/cm2,were and multiplication of PWN populations are generally cut and placed individually inside the PDA petri plates carried out in fungal cultures of Botrytis cinerea Pers., (90 mm in diameter), which had previously been incu- allowing for nematodes to multiply and complete their bated and covered entirely by the fungus. life cycle. Nematodes have several reproductive strategies, al- Nematode storage though, in this particular case, the PWN reproduces Once the population had multiplied in these plates, sexually, through amphimixis, and is oviparous, both in the medium was washed with distilled water to drag the natural and laboratory conditions (Hasegawa and nematodes toward the petri plates, which were then Miwa, 2008). Intrauterine egg development and hatch- stored in a refrigerator at 78C to maintain and preserve ing are very common in many free-living and parasitic them for 3–4 mon. The plates were examined monthly rhabditids, being more frequent in entomopathogenic to control the viability of the stored population. nematodes and unlikely in plant-parasitic nematodes, in Whenever a high mortality rate was observed, the which case it is considered a rare phenomenon (Baliadi breeding and multiplication process was restarted. et al., 2001). The purpose of this report is to describe, for the first time ever, a case of matricidal hatching in females of RESULTS AND DISCUSSION PWN reared under laboratory conditions. During the control of the viability of the stored population, in which mobility and mortality were ex- MATERIALS AND METHODS amined, a very unusual phenomenon of intrauterine egg development and hatching was recorded in B. xy- Ever since the PWN was first identified in the au- lophilus. First, a live gravid female containing three tonomous community of Galicia (northwestern Spain) unhatched eggs at different embryonic stages was ob- at the end of 2010, its population has been bred and served. One of these eggs was in an early embryonic multiplied to conduct future research on this patho- stage and the other two were in a J2 juvenile stage (Fig. gen. After testing different already-described methods 1A). This phenomenon was later observed again in four allowing for the routine maintenance and multiplication females (only one being alive); one of the females contained an unhatched egg and the other three had Received for publication June 28, 2017. Estacion Fitopatoloxica Areeiro, Diputacion de Pontevedra, E-36153 Pontevedra, hatched juveniles, which were actively moving inside Spain. the maternal body (Fig. 1C). The live female had dis- The authors thank Dr. Manuel Mota for his comments and suggestions. E-mail: [email protected]. organized inner organs and the dead females showed This paper was edited by David Shapiro-Ilan. different levels of degradation (Fig. 1B–D), with the 390 Matricidal Hatching in Pinewood Nematode: Abelleira et al. 391 FIG. 1. Different Bursaphelenchus xylophilus females with matricidal hatching. A. Detail of the vulval region of a B. xylophilus female containing three eggs in different embryonic stages and without hatching (both female and juvenile were alive before photographed). B. Vulval region of a B. xylophilus female with a juvenile J2 (both were alive before photographed). C. B. xylophilus female with a juvenile J2 in the posterior region. D. Detail of the posterior part of a B. xylophilus female of the previous image. stylet and the median bulb only remaining visible in species of other genera, as shown in Table 1. Specifically one of them. in the case of the Bursaphelenchus genus, and according The phenomenon of intrauterine egg development to the available literature, this phenomenon was only and hatching, leading to the destruction of female recorded in Bursaphelenchus eremus (Ruhm)€ Goodey nematodes by their juvenile stages (Maupas, 1899), was (Carletti et al., 2007). In this case, an embrionated egg erroneously termed endotokia matricida (endo [Greek] = was observed within the postuterine branch of a female inside, -tokia [Greek] = birth, matri [Latin] = mother, -cida nematode that had been reared on cultures of B. cinerea [Latin] = kill) by several authors, such as Southey (1969) and previously isolated from Quercus robur L. or Laughlin et al. (1978). In an attempt to clarify the Matricidal hatching in plant-parasitic nematodes is term, Luc et al. (1979) remarked the differences and a very unusual phenomenon, mainly observed in rou- suggested the use of matricidal hatching to refer to the tine analyses carried out under natural conditions, intrauterine development and hatching of eggs, leaving there being no available references on the culture the term endotokia matricida, defined by Seurat (1914), as conditions involved in the process. Previous studies of a synonym of female encystment. matricidal hatching in nonplant-parasitic nematodes, The first mention of matricidal hatching in plant- mainly in entomopathogenic and free-living nema- parasitic nematodes took place in the description of todes, such as Caenorhabditis elegans Maupas, have cited Anguina tritici Steinbuch by Scopoli in 1777 (cited as several factors that could favor the incidence and de- a female ‘‘vivipara & ovipara’’). Over the last few de- velopment of matricidal hatching. Laughlin et al. (1978) cades, this phenomenon has been reported in a few and Singh and Khera (1976) gathered the opinions of 392 TABLE 1. Nematodes reported with intrauterine egg development and hatching. Journal of Nematology, Volume 49, No. 4, December 2017 Taxonomy classification Plant parasitic nematodes Other nematodes Class Chromadorea Order Suborder Family Cephalobiidae Acrobeloides sp. (Singh and Khera, 1976) Rhabditida Rhabditina Chiloplacus lentus (Roy, 1974) Metacrobeles amblyurus (Chiu et al., 2002) Family Heterorhabditidae Heterorhabditis bacteriophora (Poinar, 1975) Heterorhabditis megidis (Ehlers et al., 1998) Heterorhabditis sp. (Johnigk and Ehlers, 1999) Family Mehdinematidae Mehdinema alii (Luong et al., 1999) Family Neodiplogasteridae Pristionchus pacificus (Gilarte et al., 2015) Family Rhabditidae Caenorhabditis elegans (Mitchell et al., 1979) Oscheius chongmingensis (Liu et al., 2012) Rhabditis terricola (Perez, 1866) Rhabditis tipulae (Lam and Webster, 1971) Rhabditis tripartita (Craveiro, 1985) Rhabditis sp. (Lordello, 1951) Suborder Family Anguinidae Anguina spp. (Ivanova, 1962) Tylenchina Anguina sp. (Southey, 1969) Anguina pacificae (McClure et al., 2008) Anguina tritici (Scopoli, 1777; Marcinowski, 1909; Gupta and Swarup, 1968) Ditylenchus destrutor (Duggan and Moore, 1962) Ditylenchus dipsaci (Teploukhova, 1968) Paranguina agropyri (Krall, 1967) Family Aphelenchoididae Aphelenchoides fragariae (Loof, 1959) Aphelenchus avenae (Jairajpuri, 1964 [unpublised, Hechler]) Bursaphelenchus eremus (Carletti et al., 2007) Bursaphelenchus xylophilus (this work) Family Brevibuccidae Tarantobelus arachnicida* (Abolafia and Pena-Santiago,~ 2017) Family Hoplolaimidae Helicotylenchus sp. (Yuen, 1965) Thecavermiculatus gracililancea (Robbins, 1978) Helicotylenchus paxilli (Yuen, 1965) Helicotylenchus vulgaris (Yuen, 1966) Family Meloidogynidae Meloidogyne sp. (Atkinson, 1889; Nagakura, 1930; Pinochet, 1978) Meloidogyne incognita (Perlaza