Procambarus Clarkii Research Articles

Effects of Different Salinity Stress on the Transcriptomic Responses of Freshwater Crayfish (Procambarus clarkii, Girard, 1852).

Salinization of freshwater ecosystems is a pressing global issue. Changes in salinity can exert severe pressure on aquatic animals and jeopardize their survival. Procambarus clarkii is a valuable freshwater aquaculture species that exhibits some degree of salinity tolerance, making it an excellent research model for freshwater aquaculture species facing salinity stress. In the present study, crayfish were exposed to acute low salt (6 ppt) and high salt (18 ppt) conditions. The organisms were continuously monitored at 6, 24, and 72 h using RNA-Seq to investigate the mechanisms of salt stress resistance. Transcriptome analysis revealed that the crayfish responded to salinity stress with numerous differentially expressed genes, and most of different expression genes was observed in high salinity group for 24h. GO and KEGG enrichment analyses indicated that metabolic pathways were the primary response pathways in crayfish under salinity stress. This suggests that crayfish may use metabolic pathways to compensate for energy loss caused by osmotic stress. Furthermore, gene expression analysis revealed the differential expression of immune and antioxidant-related pathway genes under salinity stress, implying that salinity stress induces immune disorders in crayfish. More genes related to cell proliferation, differentiation, and apoptosis, such as the Foxo, Wnt, Hippo, and Notch signaling pathways, responded to high-salinity stress. This suggests that regulating the cellular replication cycle and accelerating apoptosis may be necessary for crayfish to cope with high-salinity stress. Additionally, we identified 36 solute carrier family (SLC) genes related to ion transport, depicting possible ion exchange mechanisms in crayfish under salinity stress. These findings aimed to establish a foundation for understanding crustacean responses to salinity stress and their osmoregulatory mechanisms.

The identification, evolutionary analysis, and immune roles of Rab family members in red swamp crayfish, Procambarus clarkii

The Rab GTPase constitutes the largest family of small GTPases that regulate intracellular trafficking. Different eukaryotes possess varying numbers of Rab paralogs. However, limited knowledge exists regarding the evolutionary pattern of Rab family in most major eukaryotic supergroups. This study cloned 24 Rab genes from transcriptome data of Procambarus clarkii haemocytes. The multiple sequence alignment and phylogenetic tree analysis revealed a relatively high degree of conservation for PcRab. Furthermore, PcRab exhibited similarities in motif composition with all members showing presence of G, PM, RabF, and RabSF motifs. The tertiary structure indicated that PcRab proteins mainly consisted of α-helices and β-strands, and most PcRab proteins shared similar tertiary structures, and it was indicated that they have similar protein characteristics. Protein-protein interaction prediction identified a total of 20 interacting proteins involved in vesicle trafficking, phagocytosis, and signal transduction with 193 interactions. Expression analysis showed wide expression patterns for PcRab in P. clarkii organs. Upon infection by white spot syndrome virus and Aeromonas veronii, significant induction was observed for PcRab gene expression levels, indicating their involvement in pathogen response mechanisms. The present study represents the pioneering effort in comprehensively identifying and cloning the Rab family genes in crustacean, followed by a systematic investigation into their evolutionary patterns and immune response upon pathogen infection. The results provided valuable insights for further investigation into the molecular mechanism underlying the response of P. clarkii to pathogen infection.

Contamination Profiles of Selected Pollutants in Procambarus clarkii Non-Edible Portions Highlight Their Potential Exploitation Applications.

Properly managing aquatic organisms is crucial, including protecting endemic species and controlling invasive species. From a circular economy perspective, the sustainable use of aquatic species as a source of bioactive molecules is an area that is increasingly being explored. This includes the use of non-edible portions of seafood, which could pose considerable risks to the environment due to current methods of disposal. Therefore, it is of paramount importance to ensure that the exploitation of these resources does not result in the transfer of pollutants to the final product. This study analyzed two types of non-edible parts from the crayfish Procambarus clarkii: the abdominal portion of the exoskeleton (AbE) and the whole exoskeleton (WE), including the cephalothorax. These portions could potentially be utilized in the context of eradication activities regulated by local authorities. A screening analysis of four classes of pollutants, including pesticides, per- and polyfluoroalkyl substances (PFAS), phthalic acid esters (PAEs), and trace elements (TEs), was performed. The only analytes detected were TEs, and significant differences in the contamination profile were found between AbE and WE. Nevertheless, the levels recorded were comparable to or lower than those reported in the literature and below the maximum levels allowed in the current European legislation for food, suggesting that their potential use is legally permitted. In terms of scalability, the utilization of the entire non-edible P. clarkii portion would represent a sustainable solution for the reuse of waste products.

Effects of nitrite exposure on the oxidative stress, immune response and intestine microbiota of Procambarus clarkii

Nitrite (NIT) is a toxic nitrogenous compound that adversely affects living organisms. This study utilized pathological, metagenomic, and transcriptomic analyses to examine the toxic effects and damage mechanisms of NIT exposure on the intestine of Procambarus clarkii. Findings revealed that excessive NIT led to pathological changes in the crayfish intestine, an imbalance in intestinal flora, and an increase in harmful bacteria such as Enterobacteriaceae, Shewanella, and Vibrionaceae. Additionally, NIT exposure caused various physiological changes, including oxidative stress, immune imbalance, metabolic disorders, excessive autophagy, and apoptosis, leading to inflammatory damage in the intestine. Moreover, NIT exposure disrupted crayfish energy metabolism by reducing ATP content and disturbing energy homeostasis. The inhibition of the intestinal immune system is speculated to disrupt intestinal flora homeostasis and promote the overgrowth of opportunistic pathogenic bacteria, increasing the risk of crayfish infections. This study provides new insights into the antioxidant and immune defense mechanisms against NIT in crayfish and may aid in developing strategies to enhance disease and stress resistance in crustaceans.

Effect of abamectin on osmoregulation in red swamp crayfish (Procambarus clarkii).

As a widely used pesticide, abamectin could be a threat to nontarget organisms. In this study, the toxic mechanism of abamectin on osmoregulation in Procambarus clarkii was explored for the first time. The results of this study showed that with increasing abamectin concentration, the membrane structures of gill filaments were damaged, with changes in ATPase activities, transporter contents, biogenic amine contents, and gene expression levels. The results of this study indicated that at 0.2mg/L abamectin, ion diffusion could maintain osmoregulation. At 0.4mg/L abamectin, passive transport was inhibited due to damage to the membrane structures of gill filaments, and active transport needed to be enhanced for osmoregulation. At 0.6mg/L abamectin, the membrane structures of gill filaments were seriously damaged, and the expression level of osmoregulation-related genes decreased, but the organisms were still mobilizing various transporters, ATPases, and biogenic amines to address abamectin stress. This study provided a theoretical basis for further study of the effects of contaminations in aquatic environment on the health of crustaceans.

Prevalence of the crayfish plague pathogen in red swamp crayfish populations in western France: How serious is the risk for the native white-clawed crayfish?

The crayfish plague pathogen Aphanomyces astaci has been implicated in a number of mass mortalities and irreversible population declines of native crayfish across Europe. At present, the reservoirs of the pathogen in Europe are mainly populations of invasive North American crayfish species. In southwestern Europe, including France, a particularly widespread invader is the red swamp crayfish Procambarus clarkii. Recent distribution data confirm that P. clarkii is present in at least 75 French departments, i.e. more than 78% of those in metropolitan France. We analysed the prevalence and pathogen load of A. astaci in 42 populations of this species in western France (Nouvelle Aquitaine region), where the species is most densely distributed, particularly in a wide range of environments around the Gironde estuary. The pathogen was detected by two different quantitative PCR assays in more than three quarters of the populations studied (34 out of 42); 163 out of 480 analysed crayfish individuals tested positive for the presence of A. astaci. In most cases, individual infection levels were very low, detectable with quantitative PCR but not sufficient for pathogen genotyping. In seven P. clarkii individuals from four populations, however, we were able to assess A. astaci variation by microsatellite markers and sequencing of mitochondrial markers. All these host specimens carried A. astaci genotype group D, haplotype d1, which has caused the majority of crayfish plague outbreaks in neighbouring Spain. In contrast, the French outbreaks genotyped to date (including eight newly analysed in this study) were mostly caused by strains of genotype group B, specific to the signal crayfish Pacifastacus leniusculus. Haplotype d1 found in P. clarkii was involved in one of the newly characterised outbreaks. Our study confirms that P. clarkii is a potentially important reservoir of the crayfish plague pathogen in France, but not the main source of the pathogen in mass mortalities of A. pallipes, probably due to different ecological requirements of the different invasive host crayfish. However, as P. clarkii continues to spread, the threat posed by this species to native crayfish is likely to increase.

Water Physicochemical Properties and Metal Deposition in the Crayfish, Procambarus clarkii, and Sediments of Sharkia Province, Egypt, Under Water Pollution

Water Physicochemical Properties and Metal Deposition in the Crayfish, Procambarus clarkii, and Sediments of Sharkia Province, Egypt, Under Water Pollution

Study of technological aspects of Procambarus clarkii cultivation in terms of ensuring their welfare

Study of technological aspects of Procambarus clarkii cultivation in terms of ensuring their welfare

Hitting two targets with one shot on pesticide genotoxicity assessment – Identifying risk while unveiling ex vivo approach as a throughput tool in gill-breathing animals

Pesticides in the environment often compromise the ecosystem, thus requiring reliable approaches to assess their effects. Commonly used approaches, such as in vivo, come with several disadvantages, namely in the light of the 3 R’s policy. Seeking for accurate and ethical approaches, this study intended to validate the ex vivo technique as an alternative, and to assess the genotoxicity of chemically-based pesticides and a biopesticide. The ex vivo approach was applied to gill cells of Procambarus clarkii for 2, 4 and 8 h. Cell viability and DNA integrity were evaluated to determine the applicability of this approach. Crayfish gill cells only showed to be suitable for exposures of 2 h. Accordingly, genotoxicity was evaluated in gill cells exposed, for 2 h, to environmentally relevant concentrations of the chemically-based pesticides dimethoate (20 µg L−1), imazalil (160 µg L−1) and penoxsulam (23 µg L−1), as well as to the bioinsecticide Turex® (25, 50, 100, 200 and 400 µg L−1). Every chemically-based pesticide demonstrated to be genotoxic, despite not inducing oxidative DNA damage. On the other hand, Turex® showed no genotoxic effects. Overall, the ex vivo approach demonstrated to be possible and practical to implement, improving the number of outcomes with a lower number of organisms. The findings from the screening test suggest that biological pesticides may pose a lower risk to non-target organisms compared to chemically-based pesticides.

Interspecific variation in the defensive responses of brown frogs to alien predators

AbstractAlien species impact native amphibians through various direct effects, including predation, and pose a significant threat to naïve prey populations. In this study, we exposed tadpoles of three brown frog species (Rana dalmatina, Rana latastei, and Rana temporaria) to the olfactory cues of two alien predators, the pond slider (Trachemys scripta) and red swamp crayfish (Procambarus clarkii), and compared their responses to those induced by the aquatic larvae of a widespread native predator, the Southern hawker (Aeshna cyanea). We recorded two tadpole defensive behaviors, the proportion of time they were active and the number of freezing events. Both agile frog species, R. dalmatina and R. latastei, showed moderate responses to red swamp crayfish kairomones and strong responses to both odonate larvae and pond sliders. In contrast, the common frog (R. temporaria) displayed a less intense response to crayfish with respect to odonate larvae, and a negligible response to pond sliders. Long-lasting coexistence with either European pond turtles or pond sliders may explain the strength of agile frogs’ response toward the alien species; while, the historical range of the white-clawed crayfish (Austropotamobius pallipes) overlapped that of the common frog, enhancing the co-option of alien crayfish cues by this anuran species.

The molecular characterization of Rab11 and its immune roles in red swamp crayfish (Procambarus clarkii)

The Rab proteins primarily regulate vesicular transport between membrane-bound organelles and are important for innate immune. However, there is currently a lack of studies on crustaceans regarding Rab proteins, particularly core Rabs. We identified a Rab11 gene from Procambarus clarkii (PcRab11) and evaluated its potential involvement in immune response. The results showed PcRab11 was 1789 bp long, with an open reading frame of 645 bp encoding 211 amino acids and an estimated molecular weight of 23.8 kDa. Sequence analysis revealed its remarkable evolutionary conservation. The PcRab11 was widely expressed in various tissues, with highest levels in hepatopancreas, and localized within the cell cytoplasm. Upon infection with white spot syndrome virus (WSSV) or Aeromonas veronii, the expression of PcRab11 in immune organs was significantly induced. Furthermore, silencing PcRab11 reduced phagocytosis-related genes expression and haemocytes' phagocytic activity to FITC-labeled A. veronii, as well as decreased mortality and death time in WSSV or A. veronii infected P. clarkii. Additionally, the potential protein interaction between PcRab11 and 14-3-3ε was identified in haemocytes. Overall, our findings provided evidence for the involvement of Rab11 in P. clarkii's immune response, establishing a foundation to explore the immune role of core Rab proteins in crustaceans' innate immune system.

Longitudinal tracking of hemocyte populations in vivo indicates lineage relationships and supports neural progenitor identity in adult neurogenesis

Adult neurogenesis, which takes place in both vertebrate and invertebrate species, is the process by which new neurons are born and integrated into existing functional neural circuits, long after embryonic development. Most studies in mammals suggest that self-renewing stem cells are the source of the new neurons, although the extent of self-renewal is a matter of debate. In contrast, research in the crayfish Procambarus clarkii has demonstrated that the neural progenitors producing adult-born neurons are capable of both self-renewing and consuming (non-self-renewing) divisions. However, self-renewing divisions are relatively rare, and therefore the production of adult-born neurons depends heavily on progenitors that are not replenishing themselves. Because the small pool of neural progenitors in the neurogenic niche is never exhausted throughout the long lives of these animals, we hypothesized that there must also be an extrinsic source of these cells. It was subsequently demonstrated that the neural progenitors originate in hemocytes (blood cells) produced by the immune system that travel in the circulation before ultimately integrating into niches where the neural lineage begins. The current study examines the developmental lineage of the three hemocyte types — hyaline (HC), semigranular (SGC) and granular (GC) cells — with the goal of understanding the origins of the progenitor cells that produce adult-born neurons. Longstanding qualitative metrics for hemocyte classification were validated quantitatively. Then, in a longitudinal study, proliferation markers were used to label the hemocytes in vivo, followed by sampling the circulating hemocyte population over the course of two months. Hemolymph samples were taken at intervals to track the frequencies of the different hemocyte types. These data reveal sequential peaks in the relative frequencies of HCs, SGCs and GCs, which were identified using qualitative and quantitative measures. These findings suggest that the three hemocyte types comprise a single cellular lineage that occurs in the circulation, with each type as a sequential progressive stage in hemocyte maturation beginning with HCs and ending with GCs. When combined with previously published data, this timeline provides additional evidence that HCs serve as the primary neural progenitor during adult neurogenesis in P. clarkii.

Tandem Mass Tag-based proteomic analysis of protein changes in superchilled crayfish (Procambarus clarkii) presoaked with carrageenan oligosaccharides

To assess the effectiveness of carrageenan oligosaccharides (COs) in enhancing superchilling storage of crayfish, the physicochemical features of muscle and protein abundance in the refrigerated sample (RS), superchilled sample (SS) and COs soaked superchilled sample (CS) were evaluated. Microstructural and SDS-PAGE analyses suggested that CS exhibited fewer pores, with a microstructure and protein subunits distribution more similar to RS. Tandem Mass Tags quantitative proteomic analysis revealed 66 up-regulated differentially abundant proteins (DAPs) in the CS vs. SS batch, including myosin light chain 2, neural cadherin, integrin beta, lectin-like protein, toll-1, reticulon-1, and moesin/ezrin/radixin homolog 1, which facilitate cells adhesion and maintain membrane/cytoskeleton integrity. Eukaryotic Clusters of Orthologous Groups results confirmed that COs treatment increased the stability of crayfish myofibrillar proteins by up-regulating DAPs, which were concentrated in functional categories such as “posttranslation modification, protein turnover, chaperones”, “signal transduction mechanisms”, “energy production and conversion”, and “cytoskeleton”.

Variation of the stable isotope niches of native amphibians in ponds invaded by the red swamp crayfish

Variation of the stable isotope niches of native amphibians in ponds invaded by the red swamp crayfish

Per- and polyfluoroalkyl substances in Chinese commercially available red swamp crayfish (Procambarus clarkii): Implications for human exposure and health risk assessment

The extensive utilization of per- and polyfluoroalkyl substances (PFASs) has led to their pervasive presence in the environment, resulting in contamination of aquatic products. Prolonged exposure to PFASs has been linked to direct hepatic and renal damage, along with the induction of oxidative stress, contributing to a spectrum of chronic ailments. Despite the recent surge in popularity of red swamp crayfish as a culinary delicacy in China, studies addressing PFASs' exposure and associated health risks from their consumption remain scarce. To address this gap, our study investigated the PFASs’ content in 85 paired edible tissue samples sourced from the five primary red swamp crayfish breeding provinces in China. The health risks associated with dietary exposure were also assessed. Our findings revealed widespread detection of PFASs in crayfish samples, with short-chain perfluoroalkyl carboxylic acids (PFCAs) exhibiting the highest concentrations. Notably, the total PFAS concentration in the hepatopancreas (median: 160 ng/g) significantly exceeded that in muscle tissue (5.95 ng/g), as did the concentration of every single substance. The hazard quotient of perfluorohexanesulfonic acid (PFHxS) via consuming crayfish during peak season exceeded 1. In this case, a potential total non-cancer health risk of PFASs, which is mainly from the hepatopancreas and associated with PFHxS, is also observed (hazard index>1). Thus, it is recommended to avoid consuming the hepatopancreas of red swamp crayfish. Greater attention should be paid to governance technology innovation and regulatory measure strengthening for short-chain PFASs.

Changes in secondary sexual characteristics of female red swamp crayfish (Procambarus clarkii) and relationship to ovarian development: Implications for intensive breeding of seedlings

A rapid selection method of mature Procambarus clarkii females for use in intensive breeding of seedlings is proposed based on evaluation of secondary sexual characteristics and variation patterns in this study. Following regular sampling in ponds, the weight, cephalothorax length, annulus ventralis length, body width, white patch coverage (WC) on the uropod, and uropod content weight-to-weight (WW) ratio of female P. clarkii were found to change significantly during ovarian development. The random forest model identified white patches on the uropod as secondary sexual characteristics of female P. clarkii. In fact, the ovarian maturity status could be determined based on the variable pattern of these white patches (distribution shape, coverage, and density), particularly WC. White patches waxed and waned during ovarian development and oviposition. Specifically, white patches were absent from the uropod of female P. clarkii before the avitellogenic stage; visible on the uropod in the early vitellogenic stage (WC < 30%); WC 30–45% occurred in the midvitellogenic stage; WC > 45% occurred in the late vitellogenic stage; and white patches disappeared from the uropod of female P. clarkii in the post-vitellogenic and resorptive stage. The accuracy of identifying mature female P. clarkii (late vitellogenic stage) based on the white patch pattern was 86.36%. Hence, the observation of white patch changes on the uropod might represent an effective method for rapidly identifying mature female P. clarkii with the potential to improve artificial intensive breeding.

Population-specific responses to pollution exposure suggest local adaptation of invasive red swamp crayfish Procambarus clarkii along the Mediterranean French coastline

Anthropogenic stressors can have an impact in a broad range of physiological processes and can be a major selective force leading to rapid evolution and local population adaptation. In this study, three populations of the invasive crayfish Procambarus clarkii were investigated. They are geographically separated for at least 20 years, and live in different abiotic environments: a freshwater inland lake (Salagou lake) with no major anthropogenic influence and two other coastal wetlands regularly polluted by pesticides along the Mediterranean coast (Camargue region and Bages-Sigean lagoon). Collected adults were genetically characterized using the mitochondrial COI gene and haplotype frequencies were analyzed for genetic variability within and between populations. Results revealed a higher genetic diversity for these invasive populations than any previous report in France, with more than seven different haplotypes in a single population. The contrasting genetic diversity between the Camargue and the other two populations suggest different times and sources of introduction. To identify differences in key physiological responses between these populations, individuals from each population were maintained in controlled conditions. Data on oxygen consumption rates indicate that the Salagou and Bages-Sigean populations possess a high inter-individual variability compared to the Camargue population. The low individual variability of oxygen consumption and low genetic diversity suggest a specific local adaptation for the Camargue population. Population-specific responses were identified when individuals were exposed to a pesticide cocktail containing azoxystrobin and oxadiazon at sublethal concentrations. The Salagou population was the only one with altered hydro-osmotic balance due to pollutant exposure and a change in protease activity in the hepatopancreas. These results revealed different phenotypic responses suggesting local adaptations at the population level.

A study on the effects of replacing fishmeal with soybean meal in the feed of Procambarus clarkii: Assessing growth performance, immunity, and gut microbiota

The unstable supply and high cost of fishmeal have led to increased interest in low fish meal feed. As such, the present study aimed to investigate the effects of replacing fishmeal in feeds with soybean meal (SM) on the growth, immunity, and gut microbiota of crayfish. A total of 750 crayfish (4.00 g) were randomly divided into five groups (SS0, SS25, SS50, SS75, and SS100) with three replicates, and were fed diets with different levels of soybean meal substituted for fishmeal for six weeks. The results revealed that crayfish subjected to high SM diets experienced a significant decrease in the final body weight, weight gain rate, and specific growth rate (p<0.05), together with enzyme activities related to nutrient digestion and absorption. Of note, the groups with 25–50 % SM replacement exhibited decreased final body weight without significance compared to that in the SS0 group (p>0.05). Moreover, increasing SM inclusion exhibited the potential to impair the intestine, characterized by the increase in hemolymph LD content, decrease in the levels of TC content, T-AOC, and AKP, along with the observed edema of the lamina propria, and up-regulated mRNA expression levels of nfkb, alf, and tlr. Furthermore, high SM diets resulted in the emergence of new species and a shift in Firmicutes, Bacteroidota, Proteobacteria, and Fusobacteriota. Specifically, the abundance of lower Firmicutes and higher Proteobacteria was observed in the SS100 group, which was closely correlated with genus-level Candidatus Bacilloplasma, and RsaHf231 abundance alterations. MENAs demonstrated that the high SM diets led to a more complex, yet fragile and unstable microbial network. The global network further indicated that the microbes contributing more to crayfish were in module eigen 4, in which 9 OTUs were identified as major biomarkers and mainly classified into Proteobacteria. Taken above, SM could replace 25 % of fishmeal in crayfish diets without negatively affecting growth performance, nutrient utilization, immunity, and antioxidant capacity. Excessive SM levels contributed to gut dysfunction and weakened the immune system of crayfish.

Optimum stocking density of adult freshwater crayfish Procambarus clarkii in recirculation system

Optimum stocking density of adult freshwater crayfish Procambarus clarkii in recirculation system

Effects of Different Cultivation Modes on Morphological Traits and Correlations between Traits and Body Mass of Crayfish (Procambarus clarkii).

In this study, juvenile crayfish hatched from the same population were cultured in different growing environments: pond (D1), paddy field (D2), and aquaculture barrel (D3), and fed for 60 days. Crayfishes were selected randomly, females and males, 50 tails each from six groups (D1-♀, D1-♂, D2-♀, D2-♂, D3-♀, D3-♂) to measure the following morphological traits: full length (X1), body length (X2), chelicerae length (X3), chelicerae weight (X4), cephalothorax length (X5), cephalothorax width (X6), cephalothorax height (X7), eye spacing (X8), caudal peduncle length (X9), and caudal peduncle weight (X10). We found that the coefficient of variation (CV) of X4 was the largest in each culture mode, and males (28.58%~38.67%) were larger than females (37.76%~66.74%). The CV of X4 of crayfish cultured in D1 and D2 was larger than that of D3. All traits except X8 were positively correlated with body weight (p < 0.05). After pathway analysis, we found that X4, X5, X7, and X10 were significantly correlated with the body weight of D1-♀; the equation was YD1-♀ = -29.803 + 1.249X4 + 0.505X5 + 0.701X7 + 1.483X10 (R2 = 0.947). However, X2, X4, and X6 were significantly correlated with the body weight of D1-♂; the equation was YD1-♂ = -40.881 + 0.39X2 + 0.845X4 + 1.142X6 (R2 = 0.927). In D2-♀, X1, X4, X5, and X10 were significantly correlated with body weight; the equation was YD2-♀ = -12.248 + 0.088X1 + 1.098X4 + 0.275X5 + 0.904X10 (R2 = 0.977). X4 and X5 played a major role in the body weight of D2-♂ with the equation: YD2-♂ = -24.871 + 1.177X4 + 0.902X5 (R2 = 0.973). X3 and X10 mainly contributed to the body weight of D3-♀ with the equation: YD3-♀ = -22.476 + 0.432X3 + 3.153X10 (R2 = 0.976). X1 and X4 mainly contributed to the body weight of D3-♂ with the equation: YD3-♂ = -34.434 + 0.363X1 + 0.669X4 (R2 = 0.918). Comparing the pathway analysis with the gray relation analysis, we could conclude that the traits most correlated with body weight in D1-♀ were X10 and X7; in D1-♂, X6; in D2-♀, X10, X1, and X5; in D2-♂, X5; in D3-♀, X10; and in D3-♂, X4 and X1.