Resistance and tolerance to mixed nematode infections in relation to performance level in laying hens.

Modern chickens have been genetically developed to perform high under optimal conditions. We hypothesized that high-performance is associated with a higher sensitivity to environmental challenges in laying hens. By using nematode infections as an environmental stressor, we assessed performance-level associated host responses in a high (i.e. Lohmann Brown Plus, LB) and in a lower performing, a so-called dual-purpose chicken genotype (i.e. Lohmann Dual, LD). The hens were infected with 1000 eggs of Ascaridia galli and Heterakis gallinarum at 24 weeks of age. Hen performance parameters, humoral immune responses in plasma and egg yolks and worm burdens were assessed at several occasions over a period of 18 weeks post infection (wpi). While infections had no significant effect on feed intake (P = 0.130) and body weight in both genotypes (P = 0.392), feed conversion efficiency was negatively affected by infections (P = 0.017). Infections reduced both laying rate and egg weight and thereby per capita egg mass in both genotypes (P < 0.05). While laying rate in infected LB hens decreased significantly (P < 0.05) in the early infection period (i.e. by 3 wpi), the decrease in LD hens appeared much later (i.e. by 14 wpi). Worm burdens resulting from the experimental infection were not different between the genotypes for both worm species (P > 0.05), whereas LB hens were more susceptible (P < 0.05) to re-infections than LD hens. Changes in humoral immune responses (i.e. ascarid-specific IgY antibodies in plasma and egg yolks) of the two genotypes over time reflected closely the corresponding changes in larval counts of the hens, descending from both experimental and subsequent natural infections in both genotypes. Infections caused a shift in egg size classes, leading to smaller frequency of larger eggs in both genotypes. Infections reduced egg weight (P = 0.018) and led to a reduced fat content in the egg yolks (P = 0.045). The proportion of poly-unsaturated fatty acids (PUFA), especially n-6-PUFA, was also lower in egg yolks of the infected hens (P = 0.032). We conclude that tolerance to nematode infections in laying hens is dependent on host-performance level. The impairment in host tolerance was both genotype and time dependent, likely due to differences in genetic programming for production peak and persistency of the two genotypes. The two genotypes exhibited similar levels of resistance after a fully controlled experimental infection, but the high performing hens were more susceptible to subsequent natural infections. Infections negatively affected economically important egg-quality traits, including egg weight, fat content and fatty acid profiles in egg yolks.

Distribution of cysts of Strongyluris sp. (Nematoda) in the pallial system of Achatina fulica Bowdich, 1822 from Vila Dois Rios and Vila do Abraão, Ilha Grande, Angra dos Reis, Rio de Janeiro / Distribuição de cistos de Strongyluris sp. (Nematoda) no complexo pallial de Achatina fulica Bowdich, 1822 da Vila Dois Rios e da Vila do Abraão, Ilha Grande, Angra dos Reis, Rio de Janeiro

Abstract This work aimed to assessing Strongyluris sp. cysts distribution pattern in the several inner organs from pallial system of Achatina fulica Bowdich, 1822. Also we verified if there is a relationship between the mollusk size and the number of specimens from parasites collected from two touristic villages in Ilha Grande (Angra dos Reis, Rio de Janeiro state): Vila Dois Rios (VDR) and Vila do Abraão (ABR). The samples were obtained through a field work conducted bimonthly during 2007, 2008, 2010, and 2011, at both locations. Height and width were measured from shells collected, and the all specimens were classified in different classes: class 1 - <4.0 cm, class 2 - 4.1-9.0 cm and class 3 - < 9.0 cm. After the specimens were dissected in order to find and count the number cysts in the pallial system. In specimens from both locations, the pulmonary and secondary veins showed a high number of cysts. No significance difference was found both in the abundance of cysts among the specimens in ABR (p=0.138) and VDR (p=0.181). Achatina fulica showed different intensities of cyst infection based on the size classes: the class-3 specimens, at both locations, showed the greatest cyst average (ABR Anova F= 3.8; p=0.02); (VDR T of Student T= -2.04; p=0.04). The results suggested that the highest number of cysts in the vascularized area in pallial system of A. fulica was a consequence of a greater hemolymph circulation in that area, delivering more nutrients for larvae development. We think that bigger individuals host a higher number of cysts, as they usually present a larger biomass and a larger area of the pallial system, allowing an efficient parasite colonization. Other possible explanation could be the long exposure of the molluscs of class 3 to the parasites, which allowed a longer time to the larvae to allocate themselves.

Resumo O objetivo do estudo foi analisar o padrão de distribuição dos cistos de Strongyluris sp. nos diversos órgãos do complexo pallial de Achatina fulica Bowdich, 1822 e verificar se existe relação entre o tamanho do molusco e o número de parasitos em espécimes procedentes de dois vilarejos da Ilha Grande, Vila Dois Rios (VDR) e Vila do Abraão (ABR), município de Angra dos Reis, Rio de Janeiro. As coletas foram realizadas bimestralmente nos anos de 2007, 2008, 2010 e 2011 nas duas localidades. A altura e largura das conchas foram medidas, os espécimes classificados em classe 1 - <4,0 cm; classe 2 - 4,1-9,0 cm e classe 3 - < 9,0 cm e posteriormente dissecados sob estereomicroscópio para a busca e contagem dos cistos no complexo pallial. Em ambas localidades, VDR e ABR, as veias pulmonares e secundárias apresentaram predominância de alocação dos cistos. Não foi encontrada diferença na abundância de cistos entre os espécimes de ABR (p=0,138) e VDR (p=0,181). Achatina fulica apresentou intensidades diferentes de infecção de acordo com as classes de tamanho: os espécimes pertencentes a classe 3, nas duas localidades, apresentaram a maior média de cistos visíveis (ABR Anova F= 3,8; p=0,02); (VDR T de Student T= -2,04; p=0,04). Os resultados sugerem que o maior número de cistos na região vascularizada de A. fulica foi decorrente do maior aporte de hemolinfa nesta área, proporcionando mais nutrientes para o desenvolvimento das larvas. Acreditamos que indivíduos maiores albergam um elevado número de cistos, pois, geralmente, apresentam maior biomassa e maior área da cavidade pallial, possibilitando uma eficiente colonização dos parasitos, outra causa pode ser explicada pelo maior tempo de exposição dos moluscos da classe 3 aos parasitas, que possibilitou um período maior para as larvas se alocarem.

Distribution of cysts of Strongyluris sp. (Nematoda) in the pallial system of Achatina fulica Bowdich, 1822 from Vila Dois Rios and Vila do Abraão, Ilha Grande, Angra dos Reis, Rio de Janeiro.

This work aimed to assessing Strongyluris sp. cysts distribution pattern in the several inner organs from pallial system of Achatina fulica Bowdich, 1822. Also we verified if there is a relationship between the mollusk size and the number of specimens from parasites collected from two touristic villages in Ilha Grande (Angra dos Reis, Rio de Janeiro state): Vila Dois Rios (VDR) and Vila do Abraão (ABR). The samples were obtained through a field work conducted bimonthly during 2007, 2008, 2010, and 2011, at both locations. Height and width were measured from shells collected, and the all specimens were classified in different classes: class 1 - <4.0 cm, class 2 - 4.1-9.0 cm and class 3 - < 9.0 cm. After the specimens were dissected in order to find and count the number cysts in the pallial system. In specimens from both locations, the pulmonary and secondary veins showed a high number of cysts. No significance difference was found both in the abundance of cysts among the specimens in ABR (p=0.138) and VDR (p=0.181). Achatina fulica showed different intensities of cyst infection based on the size classes: the class-3 specimens, at both locations, showed the greatest cyst average (ABR Anova F= 3.8; p=0.02); (VDR T of Student T= -2.04; p=0.04). The results suggested that the highest number of cysts in the vascularized area in pallial system of A. fulica was a consequence of a greater hemolymph circulation in that area, delivering more nutrients for larvae development. We think that bigger individuals host a higher number of cysts, as they usually present a larger biomass and a larger area of the pallial system, allowing an efficient parasite colonization. Other possible explanation could be the long exposure of the molluscs of class 3 to the parasites, which allowed a longer time to the larvae to allocate themselves.

Morphological and molecular characterization of Megalobatrachonema hainanensis sp. nov. (Nematoda: Ascaridida), with phylogenetic position of Megalobatrachonema in Cosmocercoidea.

The genus Megalobatrachonema is a rare group of nematode parasites within Ascaridida. The systematic status of Megalobatrachonema in the superfamily Cosmocercoidea (Ascaridida) has long been controversial. The relationship of Megalobatrachonema and Chabaudgolvania remains unsolved. In the present study, a new species of Megalobatrachonema, M. hainanensis sp. nov., was described based on specimens collected in Amolops hainanensis (Boulenger) and Hylarana spinulosa (Smith) (Amphibia: Anura) from Hainan Island, China. The large ribosomal DNA (28S) and internal transcribed spacer (ITS1-5.8S-ITS2) were also sequenced for molecular identification and phylogenetic study. Phylogenetic analyses using maximum likelihood (ML) inference and Bayesian inference (BI) based on 28S and ITS1 sequence data, respectively, supported that Megalobatrachonema is a member of the family Kathlaniidae. In addition, the genetic comparison and phylogenetic results based on ITS1 sequence data also supported that the genus Chabaudgolvania should be considered as a synonym of Megalobatrachonema.

A survey of nematodes of the genus Cucullanus Müller, 1777 (Nematoda, Seuratoidea) parasitic in marine fishes off Brazil, including description of three new species.

A taxonomic survey of six nematode species (including three new taxa) from the genus Cucullanus Müller, 1777, parasites of marine fishes off the Brazilian coast, is provided. Nematodes were studied using light and scanning electron microscopy (SEM). Cucullanus gastrophysi n. sp. parasitic in Lophius gastrophysus Miranda Ribeiro differs from its congeners by the combination of the following features: shape and number of sclerotized structures in the oesophastome (a pair of lateral elongate structures and a single small reniform one), position of deirids and excretory pore (both anterior to oesophagus base), spicule length and spicule/body length ratio (0.97-1.29 mm and 6.5-10.5%, respectively), morphology and length of gubernaculum (V-shaped, 107-135 µm long). Cucullanus protrudens n. sp. from Pagrus pagrus (Linnaeus) has the cloacal lips broadly protruded, which differentiates it from several species of Cucullanus; other features, e.g., the length of spicules and gubernaculum (400-415 µm and 91-103 µm, respectively), arrangement of caudal papillae and position of excretory pore (slightly posterior to oesophagus-intestine junction) also characterize this species. Cucullanus pseudopercis n. sp. from Pseudopercis semifasciata (Cuvier) has deirids and excretory pore posterior to the oesophagus-intestine junction, which distinguishes the species from most of the congeners; furthermore, the arrangement of caudal papillae in combination with the length of spicules and gubernaculum (1.0-1.5 mm and 178-196 µm, respectively) separate this species from other taxa. Newly collected specimens of C. cirratus Müller, 1777 (type species of the genus) from Urophycis brasiliensis (Kaup), C. pedroi from Conger orbignianus Valenciennes (type host of the species) and C. genypteri Sardella, Navone & Timi, 1997 from Genypterus brasiliensis Regan, were studied as well. Comparisons between newly collected samples and the taxonomic data available for each respective species revealed features that were not previously mentioned (e.g. presence of unpaired cloacal papilla, detailed morphology of cloacal lips), as well as negligible differences in morphometry and caudal papillae arrangement. Observations on the type material of C. carioca suggested affinities with the genus Dichelyne Jägerskiöld, 1902; however, the poor preservation of these specimens does not allow further conclusions. Cucullanus rougetae is considered to be a species inquirenda.

New species of Cucullanus (Nematoda: Cucullanidae), an intestinal parasite of the peacock bass Cichla piquiti (Perciformes: Cichlidae) from the Tocantins River, Brazil.

Cucullanus tucunarensis n. sp. (Cucullanidae) is described parasitizing the peacock bass Cichla piquiti (Cichlidae) from the Tocantins River, Tocantins State, Brazil. The new species is unique and differs from all its congeners by having a tail tip provided with several sclerotized spine-like processes on its ventral side, present in both the male and female. Furthermore, C. tucunarensis n. sp. is compared with other species of the genus recorded in the same zoogeographical region and in the same group of hosts (Perciformes).

The first report of new species: Trichuris landak n. sp.

OBJECTIVE: To study nematode parasites morphology of Hystrix javanica (H. javanica), both through the feces and internal organs. METHODS: Feces were observed by direct smear method, internal organs were observed after dissecting the host. Specimens for light microscopy examination were fixed with 70% warm alcohol, cleared and mounted in lactophenol for wet mounting. Specimens for SEM examination were postfixed in cacodylate buffer and glutaraldehyde, dehydrated through a graded series of alcohol and freeze dried. The specimens were attached to stubs with double cello-tape, coated with gold and observed with a JSM5310 LV electron microscope. Figures were made with the aid of a drawing tube attached to Olympus compound microscope, other figures were photographs of scanning electron microscope images. Measurements were given in micrometers as the mean followed by the range in parentheses, unless otherwise stated. RESULTS: The nematode species found in the intestine of H. javanica are Gireterakis girardi and a new species, Trihuris landak. The new species differs with previously reported species from Hystrix because of having stylet and short cervical alae. The pattern of bacillary band is closed to Trichuris trichiurus, the species that infect human, but differs because the surface of its vulva is not covered with densely spine. CONCLUSIONS: The species of nematodes found on H. javanica were Gireterakis girardi and a new species Trichuris landak n.sp. Those two species are newly recorded in Indonesia.

[Development of a new hydrocarbon extract from the medicinal raw material of Circassian walnut (Juglans regia) and study of its antiparasitic activity].

The authors developed a technology for preparing a hydrocarbon extract from the medicinal raw material of Circassian walnut (Juglans regia), including its green fruits, green leaves, and fresh roots. To prepare the preparation, they obtained for the first time a new extragent called petroleum Russia that was found to contain more than hundred chemical compounds by chromatography mass spectrometry. The new agent was named irillen. Experiments on albino mice and albino rats established that the new agent was low toxic. The lethal doses of irillen were calculated: LD50 was 16377 +/- 457.5 mg/kg; LD16 = 12986.4 mg/kg; LD84 was 18976.6 mg/kg for albino mice; LD50 was 16998.0 +/- 535.4 mg/kg; LD16 = 12875.3 mg/ kg; LD84 = 18583.4 mg/kg for albino rats. The irillen prepared by the authors should be referred to as a low toxic and practically nontoxic agent (Toxicity Class IV and V). Irillen has a broad spectrum of antiparasitic activity. It is effective in treating toxocariasis in dogs, larval alveolar echinococcosis, ascaridiasis, and eimeriasis in chickens, and siphachiasis.

Development of larval Contracaecum rudolphii Hartwich, 1964 (Ascaridida: Anisakidae) in experimentally infected goldfish (Carassius auratus L., 1758).

Laboratory-bred goldfish were experimentally infected with Contracaecum rudolphii, either directly (with nematode larvae) or indirectly via infected zooplankton. Intensity and prevalence of infection were markedly higher in fish exposed to the infected zooplankton than in goldfish exposed to nematode larvae. When transmitted to fish via the zooplankton, larvae developed much faster and, after 8 weeks, became firmly encysted in the intestine wall. The digestive tract of larvae isolated from the intestine wall showed a well-developed ventriculus, ventricular appendix and intestinal caecum. The mouth was surrounded by three lips. When free-living larvae induced infection, they rapidly penetrated the intestine wall and migrated to internal organs (e.g. liver). The primordial lips of those larvae were poorly visible, the ventricular appendix was short, and the intestinal caecum was absent. During the 10-week experiment, no encysted larvae were found in fish infected with free-living larvae.

Cathepsin B- and L-like cysteine protease activities during the in vitro development of Hysterothylacium aduncum (Nematoda: Anisakidae), a worldwide fish parasite.

Proteinases play an important role as virulence factors both in the life-cycle of parasites and in the pathogen-host relationship. Hysterothylacium aduncum is a worldwide fish parasite nematode which has been associated with non-invasive anisakidosis and allergic responses to fish consumption in humans. Cysteine proteinases have been associated with allergy to plant pollens, detergents and dust mites. In this study the presence of two types of cysteine proteinases (cathepsin B and cathepsin L) during in vitro development of H. aduncum is investigated. Specific fluorescent substrates were used to determine cathepsin activities. The activity detected with substrate Z-FR-AMC was identified as cathepsin L (optimum pH=5.5; range 3.5-6.5). Cathepsin B activity was only identified with Z-RR-AMC (optimum pH=7.0-7.5; range 5.0-8.0). The start of cultivation led to increased activity of both cathepsins (1.8-fold for cathepsin B and 6.3-fold for cathepsin L). These activities varied according to the developmental stage. Cathepsin B activity decreased after M4, returning to its initial level. Cathepsin L activity also decreased after M4, but still maintained a high level (4-6 times the initial level) in adult stages. Having considered these activity variations and the optimum pH values, we suggest that cathepsin L has a role in digestive processes while cathepsin B could be involved in cuticle renewal, among other possible functions.

Terranova galeocerdonis (Thwaite, 1927) (Nematoda: Anisakidae) from Carcharias taurus (Chondrichthyes: Odontaspididae) off Argentina, with comments on some related species.

The purpose of this study was to study and redescribe Terranova galeocerdonis (Thwaite, 1927) from Carcharias taurus off Argentina. Its fourth larval stage was described, measured and illustrated for the first time. The host and geographical range of this species is extended into the southwestern Atlantic Ocean. When the present material was compared with T. rochalimai (Pereira, 1935) and T. ginglymostomae Olsen, 1952, it was considered that both morphological and morphometrical differences between the three species did not justify differentiation at the specific level. As a result, T. rochalimai and T. ginglymostomae are considered to be junior synonyms of T. galeocerdonis.

Survey on helminthofauna in pheasants from Eastern Europe.

In order to assess the parasitological status in imported pheasants, 51 birds (Phasianus colchicus) coming from Poland and Rumania and used in Italy in repopulation interventions for hunting purposes were examined. From each animal the trachea, oesophagus, crop and intestine were collected and examined for the presence of nematodes. The examination of the oesophagi and crops of 5 birds revealed the presence of parasites pertaining to the family Capillariidae: Eucoleus contortus was found in all of the 5 animals, E. annulatus was present along with the previous parasite in one animal coming from Poland. The examination of the tracheae revealed the presence of Syngamus trachea in 5 animals (9.80%). Adult or larval stages of Heterakis gallinarum (37.25% of birds) and Capillariidae (35.29%) were found in the intestinal tracts. Aonchotheca caudinflata was detected only in one bird coming from Poland; the capillarids found in all of the remaining pheasants exhibited morphological characteristics referable to Capillaria phasianina, a species never reported in Italy. The release of game from foreign countries, therefore, may always constitute a risk for the autochthonous one due to the spread of new parasitic infections.

Stochasticity accelerates nematode egg development.

Day-degrees models of nematode development assume that temperature stochasticity has no effect on the development rate of infective stages as long as the mean temperature is held constant. This assumption was tested in this study. Unembryonated Heterakis gallinarum eggs were subjected to nocturnal and diurnal daily temperature cycles at 12 and 17 C. respectively, and embryonation was compared with eggs subjected to similar stochastic daily cycles, in which random normal variations in the temperature were added to the 2 temperatures. The prediction that there is no effect of stochasticity was refuted. Embryonation of eggs subjected to variable daily cycles occurred significantly earlier than that of eggs subjected to deterministic daily cycles, suggesting that stochastic variation in temperature accelerated embryonation even though mean temperatures were the same. These findings show that the development time of H. gallnarum eggs is decreased by stochastic variation in temperature, which may have important implications for the effects of climate change on parasite availability.

Regulation of nematode fecundity in the ring-necked pheasant (Phasianus colchicus): not just density dependence.

Patterns of nematode fecundity were investigated for infections of the caecal worm Heterakis gallinarum in the ring-necked pheasant (Phasianus colchicus). Worm length was a good predictor of parasite fecundity. After controlling for worm length no other factors, including parasite intensity, were related to worm fecundity. Density dependence in worm size was detected in natural infections at parasite intensities above a threshold of 96 worms (worm size decreased with increasing parasite intensity). However, below this threshold, worm size actually decreased with decreasing parasite intensity (inverse density dependence). The interaction between density dependence and inverse density dependence in regulating the development and subsequent fecundity of H. gallinarum worms in ring-necked pheasants was demonstrated in an infection experiment. Density dependence was observed in the stunted growth of worms in heavily infected hosts, relative to worms in lightly infected hosts. Inverse density dependence in worm size was the common pattern across hosts by the end of the experiment, when parasite intensities were below the density dependence threshold. This is the first study to document both density dependence and inverse density dependence in parasite fecundity in the same host-helminth system.

[Preliminary study of the larvae (Nematode: Ascaridida) Paralichthys orbignyyanus parasite (Valenciennes, 1839) and Paralichthys patagonicus (Pisces: Pleuronectiformes)]. / Estudio preliminar de las larvas (Nematoda: Ascaridida) parásito de Paralichthys orbignyanus (Valenciennes, 1839) y Paralichthys patagonicus (Pisces: Pleuronectiformes).

The present paper deals with a preliminary study of different larvae of nematoda found in two species of fish Paralichthys orbignyanus and Paralichthys patagonicus. In P. orbignyanus the genus Terranova type A was found. Both Anisakis type I and Hysterothylacium were present in P. patagonicus. Different types of larvae belonging to genera Terranova (B and B'), Contracaecum (A and B) and Raphidascaris occurred in both P. patagonicus and P. orbignyanus. The specimens of Nematoda were described and the corresponding morphometric indexes were determined. The abundance of Nematoda obtained in relation to the diet of the flounders led us to conclude that P. orbignyanus feeds basically on crabs and fish, while P. patagonicus has a more diverse diet.

Baylisascariasis in raccoons from southern coastal Texas.

Two hundred and two Baylisascaris procyonis were collected from 23 (70%) of 33 raccoons (Procyon lotor) at three localities in southern coastal Texas (USA). Abundances of B. procyonis were similar among collection localities. The presence of B. procyonis in Texas is confirmed, and this record considerably extends the potential range of baylisascariasis larval migrans in North America.

Effects of testosterone on Heterakis spumosa infections in mice.

This study describes the effects of testosterone (Te) on the intestinal nematode Heterakis spumosa in mice. The course of Heterakis infections is apparently under Te-control. At high circulating Te-levels as occurring in intact males, Te-treated females, and Te-treated castrated males, the period of release of Heterakis eggs in mouse faeces is greatly extended and the number of eggs released per unit time is markedly elevated in comparison to low Te-levels, as found in untreated females and castrated male mice. Also, the onset of the patent period occurs earlier in Te-treated mice. Testosterone also accelerates development and growth of both female and male worms of Heterakis in mice. Thus, young adult male worms can be observed in the upper colon of Te-treated castrated male mice on day 21 post-infection (p.i.), whereas, at that time, only L4 larvae are present in Te-untreated male castrates. Testosterone also favours the survival of nematodes in hosts. In untreated male castrates, the number of worms present on day 7 p.i. (L2 larvae) is approximately two thirds higher than that found on day 21 p.i. However, such a reduction in the number of worms does not occur in Te-treated castrated mice during the same period of time. The early phases of the life-cycle of Heterakis, i.e. hatching in the small intestine and final settling of L2 larvae in the upper colon are independent of Te. Also, Te does not affect motility and even slightly reduces the fecundity of adult female worms in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Experimental heteroxenous cycle of Lagochilascaris minor Leiper, 1909 (Nematoda: Ascarididae) in white mice and in cats.

Reports of natural infections of sylvatic carnivores by adult worms of species similar to Lagochilascaris minor in the Neotropical region led to attempts to establish experimental cycles in laboratory mice and in cats. Also, larval development was seen in the skeletal muscle of an agouti (Dasyprocta leporina) infected per os with incubated eggs of the parasite obtained from a human case. In cats, adult worms develop and fertile eggs are expelled in the feces; in mice, larval stages of the parasite develop, and are encapsulate in the skeletal muscle, and in the adipose and subcutaneous connective tissue. From our observations, we conclude that the larva infective for the mouse is the early 3rd stage, while for the final host the infective form is the later 3rd stage. A single moult was seen in the mouse, giving rise to a small population of 4th stage larvae, long after the initial infection.

The importance of sculpin (Myoxocephalus scorpius) as intermediate host and transmitter of the sealworm Pseudoterranova decipiens.

A total of 186 sculpins (Myoxocephalus scorpius) were caught at Vega, Nordland, Norway, and examined for sealworm larvae (Pseudoterranova decipiens). Over 80% of the fish were infected. Very high infections were found with a maximum of nearly 300 larvae in one sculpin. Infections of this magnitude have so far not been reported from sculpins or any other fish species of this size. The importance of the sculpin in the life cycle and in the transmission of P. decipiens to seals or other fish species is briefly discussed.